Treatment of fabrics, fibers, or yarns

ABSTRACT

The present invention relates to a method for treating textiles with a carbohydrate oxidase and/or a fatty acid oxidizing enzyme.

FIELD OF THE INVENTION

The present invention relates to a method of treating textiles, inparticular fabrics, fibers, or yarns comprising treating the fabric,fiber, or yarn in an aqueous medium with a carbohydrate oxidase and/or afatty add oxidizing enzyme. More particularly, the invention relates tousing carbohydrate oxidase in a method for bleaching textiles, inparticular fabrics, fibers, or yarn to obtain an improved level ofwhiteness. The present invention also relates to a method of treatingtextiles with a fatty acid oxidizing enzyme and the use of a fatty acidoxidizing enzyme for improving the wettability of textiles (waterabsorbance) and/or whiteness of textiles.

BACKGROUND OF THE INVENTION

Preparatory processes are necessary for removing natural and man-inducedimpurities from fibers and for improving their aesthetic appearance andprocessability prior to for instance dyeing, printing and finishing.This purification treatment is referred to as preparation. Commonpreparation processes include desizing of cotton, silk and syntheticfibers, scouring of cotton and wool, and bleaching.

Sizing may be necessary to prevent breakage and lower processing speedsof a variety of natural and synthetic fiber yarns during their weaving.Common size agents are starches (or starch derivatives and modifiedstarches), poly(vinyl alcohol), carboxyl methyl cellulose (i.e. CMC)where starches are dominant. Paraffin, acrylic binders and variety oflubricants are often included in the size mix. After the fabric is made,size on the fabric must be removed again (i.e. desizing).

Desizing is the degradation and/or removal of sizing compounds from warpyarns in a woven fabric. Starch is usually removed by an enzymaticdesizing procedure. In addition, oxidative desizing and chemicaldesizing with acids or bases are sometimes used. Typical enzymes fordesizing are alpha-amylase, beta-amylase, amyloglucosidase, or mixturesthereof (see e.g. U.S. Pat. No. 5,364,782, U.S. Pat. No. 5,769,900, U.S.Pat. No. 6,017,751). Cellulase and lipase are also used either alone orcombined with amylase for desizing (WO 96/05353, Textile Chemist andColorist 29(6), 23-26(1999)).

Scouring is used to remove impurities from the fibers, to swell thefibers and to solubilize seed coat. It is one of the most criticalsteps. The main purposes of scouring is to a) uniformly clean thefabric, b) soften the motes and other trashes, c) improve fabricabsorbency, d) saponify and solubilize fats, oils, and waxes, and e)minimize immature cotton. Sodium hydroxide scouring at about boilingtemperature Is the accepted treatment for 100% cotton, while calciumhydroxide and sodium carbonate are less frequently used. Syntheticfibers are scoured at much milder conditions. Surfactant and chelatingagents are essential for alkaline scouring (Alkaline treatment ofcellulose fibers, in Handbook of fiber Science and Technology 1(A),Textile Processing and Properties, in Textile Sciences and Technology11). Enzymatic scouring has been introduced recently (U.S. Pat. No.5,912,407, JP 51-149976, WO 98/06857, U.S. Pat. No. 6,066,494).Cellulase, hemicellulase, pectinase, lipase, and protease are allreported to have scouring effects.

Bleaching is the destruction of pigmented color and colored Impuritiesas well as seed coat fragment removal. It is the most critical chemicaltreatment since a balance between the degrees of whiteness without fiberdamage must be maintained. Bleaching is performed by the use ofoxidizing or reducing chemistry. Oxidizing agents can be furthersubdivided into those that employ or generate: a) hypochlorite (OCl⁻),b) chloride dioxide (ClO₂), and hydroperoxide species (OOH⁻ and/or OOH).Reducing agents are typical sulfur dioxide, hydrosulfite salts, etc.Enzymatic bleaching using glucose oxidase has been reported (Ishihara,et al, Enzymatic Processes for Bleaching Cotton Fabrics, Shizuoka-KenHamamatsu Kogyo Gijutsu Senia Kenkyu Hokoku 7, 7-13 (1997).Buschle-Diller and Yang, Enzymatic Bleaching of Cotton Fabric withGlucose Oxidase, Textile Res. J. 71(5), 388-394 (2001). Tzanov, et al,Bio-Preparation of Cotton Fabrics, Enzyme Microb. Technol. 29, 357-362(2001).

In industrial practice, equipment availability, fabric construction andcustomer requirements all influence the choice of processes ofpreparation. Various batch, demi-continuous, and continuous processesare used. In order to give the optimum base fabric for subsequent dyeingand finishing for production of quality products, a total engineeredstrategy for desizing, scouring and bleaching must be employed. The mostcommonly used strategy is 1) single stage preparation where desizing,scouring and bleaching are conducted in one operation, 2) three-stagepreparation where the operations are conducted in sequence asdesizing-wash-scouring-wash-bleaching-wash. While the single-stageapproach saves energy and floor space, the conventional three-stageoperation gives high quality of prepared fabrics.

Buschle-Diller et al.: Enzymatic Bleaching of Cotton Fabric with GlucoseOxidase, Textile Res. J. 71(5), 388-394 (2001) discloses that thetreatment bath from the desizing with amyloglucosidase combined withbioscouring of cotton fabric can be reused for enzymatic bleaching withglucose oxidase. The reference discloses that after generation ofperoxide by glucose oxidase in a first step, the pH was adjusted to 7and bleaching performed at 85-90° C. for 60-120 minutes in a secondstep.

Tzanov et al.: Bio-preparation of cotton fabrics, Enzyme and MicrobialTechnology 29 (2001), 357-362 discloses an enzymatic process forscouring and bleaching of cotton fabrics based on the use of pectinaseand glucose oxidase.

There is still a need for improved processes for treating textiles.

SUMMARY OF THE INVENTION

In the main aspect the invention relates to a method of treatingtextiles, In particular fabrics, fibers, or yarns comprising treatingfabric, fiber, or yarn, in an aqueous medium, with a carbohydrateoxidase and/or a fatty acid oxidizing enzyme.

In one embodiment the invention provides an enzyme-based method fortreating textiles, in particular fabrics, fibers or yarn, comprisingtreating the fabric, fiber, or yarn in an aqueous medium with acarbohydrate oxidase, and in particular to a method for bleachingfabric, fiber or yarn. Carbohydrate oxidases have activity towards aplurality of substrates, i.e., the carbohydrate oxidase has activitytowards mono-saccharides and at least one of di-saccharides andoligo-saccharides. Accordingly, although not limited to any one theoryof operation, the use of a carbohydrate oxidase in accordance with thepresent invention is advantageous in that bleaching can be carried outagainst a broad range of substrates. The bleaching process can becarried out with the choice between different substrates making themethod more applicable for the bleaching purpose as the person toconduct the bleaching process can choose from a broader range of sugarsubstrates either generated in situ with another enzyme or chemicalsystem, from starch sizing and/or cellulosic fiber or added which is notthe case when limited to a specific choice of substrate. In addition,the method can be carried out without the use of environmentallydamaging chemicals and without using large amounts of rinse water.

One embodiment of the invention provides a method of manufacturing ableached fabric, fiber or yarn comprising treating fabric, fiber or yarnin an aqueous medium with an effective amount of a carbohydrate oxidaseand a carbohydrate oxidase substrate.

In another embodiment, the present invention provides an improved methodof treating textiles with a fatty acid oxidizing enzyme. The presentinventors have found that a fatty acid oxidizing enzyme advantageouslymay be used for treatment of textiles. Thus, in this aspect theinvention relates to a method of treating textile, in particularfabrics, garments, or yarns, comprising a step of treating the textilein an aqueous medium with one or more fatty acid oxidizing enzyme.

In a third aspect the invention relates to a composition comprising afatty acid oxidizing enzyme and in addition thereto at least oneadjuvant. Examples of adjuvants, which are used for treating textiles,include wetting agents, such as certain surfactants; polymeric agents;and dispersing agents.

At least in context of the present invention the terms “method” and“process” may be used interchangeably.

DETAILED DESCRIPTION OF THE INVENTION

In the main aspect the invention relates to a method of treatingtextiles, in particular fabrics, fibers, or yarns comprising treatingfabric, fiber, or yarn, in an aqueous medium, with a carbohydrateoxidase and/or a fatty acid oxidizing enzyme.

The present invention is directed to a method for bleaching fabrics,fibers and yarns, wherein the fabrics, fibers, and yarns are treated inan aqueous medium with an effective amount of a carbohydrate oxidasehaving activity towards monosaccharides and at least one ofdi-saccharides and oligo-saccharides and a substrate for saidcarbohydrate oxidase.

The present invention also provides an improved method of treatingtextiles. The present inventor has found that a fatty acid oxidizingenzyme advantageously may be used for treatment of textiles. Theinventor found that when using a fatty acid oxidizing enzyme fortreating textiles bleaching is observed. An alkaline treatment usingsodium hydroxide (NaOH) at high temperatures (around 95° C.) furtherincreased the bleaching effect. It was also found that the presence of asubstrate to the fatty acid oxidizing enzyme (e.g., lenoleic acid) has awhitening effect on the textile. When the fatty acid oxidizing enzyme isused together with a pectolytic enzyme on a textile the fabricwettability (i.e., wetting time) is enhanced. Combining the fatty acidoxidizubg enzymes with a substrate thereto increases the whiteness ofthe textile. When a fatty acid oxidizing enzyme is used on desizedtextiles together with a lipolytic enzyme and a pectolytic enzyme thewhiteness is improved. Addition of a substrate to the fatty acidoxidizing enzyme further improves the whiteness. When using a fatty acidoxidizing enzyme alone or in combination with a substrate for desizingof textiles in the presence of an amylase or an amylase and a lipolyticenzyme the whiteness and fabric wettability is improved.

The term “textiles” used herein is meant to include fabrics, garments,or yarns.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, references to “carbohydrate oxidase” include the use of one ormore carbohydrate oxidases and references to “fatty acid oxidizingenzyme” include the use of one or more fatty acid oxidizing enzymes.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of disclosing anddescribing the material for which the reference was cited in connectionwith.

EC-numbers may be used for classification of enzymes. Reference is madeto the Recommendations (1992) of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology, AcademicPress Inc., 1992.

It is to be understood that the term enzyme, as well as the variousenzymes and enzyme classes mentioned herein, encompass wild-typeenzymes, as well as any variant thereof that retains the activity inquestion. Such variants may be produced by recombinant techniques. Thewild-type enzymes may also be produced by recombinant techniques, or byisolation and purification from the natural source. In an embodiment theenzyme in question is well-defined, meaning that only one major enzymecomponent is present. This can be inferred, e.g., by fractionation on anappropriate size-exclusion column. Such well-defined, or purified, orhighly purified, enzyme can be obtained as is known in the art and/ordescribed in publications relating to the specific enzyme in question.

Even if not specifically mentioned in connection with treatment oftextiles with (an) enzyme(s) or agent(s) according to the method of theinvention, it is to be understood that the enzyme(s) or agent(s) is(are)used in an “effective amount”. The term “effective amount” means in thecontext of the present invention an amount of carbohydrate oxidase thatis able to generate enough hydrogen peroxide to provide bleaching of thetextile material as compared to a textile material which has not beentreated with a carbohydrate oxidase. In context of, e.g., fatty acidoxidizing enzyme it means the amount of enzyme capable of providing thedesired effect, such as desizing, scouring, and/or bleaching effect onthe textile as compared to a textile which has not been treated withsaid fatty acid oxidizing enzyme.

The term “applied together with” (or “used together with”) means thatthe additional enzyme may be applied in the same, or in another step ofthe method of the invention. The other treatment step(s) in the methodof the invention may be carried out upstream or downstream in thetextile treatment method, as compared to the step in which the textileis treated with a fatty acid oxidizing enzyme.

The term “a step” of a method means at least one step, and it could beone, two, three, four, five or even more method steps. In other wordsthe fatty acid oxidizing enzyme used according to the invention may beapplied in at least one method step, and the additional enzyme(s) mayalso be applied in at least one method step, which may be the same or adifferent method step as compared to the step where the fatty acidoxidizing enzyme is used.

The term “bleaching” is here defined as a whitening of the fabric,fiber, or yarn. The value of whiteness index (WI) is measured using aMacBeth Color Eye equipped with Optiview 7000 software. The Whitenessindex is calculated from the following equation:WI=Y+800(x _(n) −x)+1700(y _(n) −y)where Y, x and y are chromaticity coordinates of the sample, and x_(n)and y_(n) are those of illuminant using the standard illuminant D65.Textiles

In context of the invention the term “textile” includes fabrics,garments, and yarns.

Fabric can be constructed from fibers by weaving, knitting or non-wovenoperations. Weaving and knitting require yarn as the input whereas thenon-woven fabric is the result of random bonding of fibers (paper can bethought of as non-woven). In the present context, the term “fabric” isalso intended to include fibers and other types of processed fabrics.

Woven fabric is constructed by weaving “filling” or weft yarns betweenwrap yarns stretched in the longitudinal direction on the loom. The wrapyarns must be sized before weaving in order to lubricate and protectthem from abrasion at the high speed insertion of the filling yarnsduring weaving. The filling yarn can be woven through the warp yarns ina “over one—under the next” fashion (plain weave) or by “over one—undertwo” (twill) or any other myriad of permutations. Strength, texture andpattern are related not only to the type/quality of the yarn but alsothe type of weave. Generally, dresses, shirts, pants, sheeting's,towels, draperies, etc. are produced from woven fabric.

Knitting is forming a fabric by joining together interlocking loops ofyarn. As opposed to weaving which is constructed from two types of yarnand has many “ends”, knitted fabric is produced from a single continuousstrand of yarn. As with weaving, there are many different ways to loopyarn together and the final fabric properties are dependent both uponthe yarn and the type of knit. Underwear, sweaters, socks, sport shirts,sweat shirts, etc. are derived from knit fabrics.

Non-woven fabrics are sheets of fabric made by bonding and/orinterlocking fibers and filaments by mechanical, thermal, chemical orsolvent mediated processes. The resultant fabric can be in the form ofweb-like structures, laminates or films. Typical examples are disposablebaby diapers, towels, wipes, surgical gowns, fibers for the“environmental friendly” fashion, filter media, bedding, roofingmaterials, backing for two-dimensional fabrics and many others.

According to the invention, the method of the invention may be appliedto any fabric known in the art (woven, knitted, or non-woven). Inparticular the bleaching process may be applied to cellulose-containingor cellulosic fabrics, such as cotton, viscose, rayon, ramie, linen,lyocell (e.g. Tencel, produced by Courtaulds Fibers), or mixturesthereof, or mixtures of any of these fibers together with syntheticfibres (e.g., polyester, polyamid, nylon) or other natural fibers suchas wool and silk., such as viscose/cotton blends, lyocell/cotton blends,viscose/wool blends, lyocell/wool blends, cotton/wool blends; flax(linen), ramie and other fabrics based on cellulose fibers, includingall blends of cellulosic fibers with other fibers such as wool,polyamide, acrylic and polyester fibers, e.g., viscose/cotton/polyesterblends, wool/cotton/polyester blends, flax/cotton blends etc. The term“wool,” means any commercially useful animal hair product, for example,wool from sheep, camel, rabbit, goat, llama, and known as merino wool,Shetland wool, cashmere wool, alpaca wool, mohair, etc. and includeswool fiber and animal hair. The method of the invention can be used withwool or animal hair material in the form of top, fiber, yarn, or wovenor knitted fabric. The enzymatic treatment can also be carried out onloose flock or on fibers made from wool or animal hair material. Thetreatment can be performed at many different stages of processing. Thefabric to be bleached may be dyed or undyed. According to the inventiontextile may be desized, scoured and/or bleached in aqueous medium in thepresence of a fatty acid oxidizing enzyme.

Mote Particles

Mote particles are dark brown particles found on unbleached cottonfabric, also called “dark spots”. They are cotton pod and stem residuesoriginating from the mechanical picking of cotton. The brown color isdue to the high lignin content of the mote particles.

Desizing

According to the invention desizing may be carried out at conditionschosen to suit the method according to principles well known in the art.In an embodiment a sized fabric in either rope or open width form isbrought in contact with the processing liquid containing a fatty acidoxidizing enzyme and desizing agents. The desizing agents employeddepend upon the type of size to be removed. The most common sizing agentis based upon starch. Therefore in a preferred embodiment the textile isdesized by a combination of hot water (i.e., 50-100° C., preferably 60°C. to 80° C.), an alpha-amylase and a wetting agent and/or surfactant.

The textile is allowed to stand with the desizing agents for a “holdingperiod” sufficiently long to accomplish the desizing. The holding periodis dependent upon the type of processing regime and the temperature andcan vary from 15 minutes to 2 hours, or in some cases, several days.Typically, the desizing agents are applied in a saturator bath whichgenerally ranges from about 15° C. to 60° C. The textile is then held inequipment such as a “J-box” which provides sufficient heat, usuallybetween 50° C. and 100° C. to enhance the activity of the desizingagents. The agents, including the removed sizing agents, are washed awayfrom the textile after the termination of the holding period.

In order to ensure a high whiteness and/or a good dyeability, the sizeand other applied agents must be thoroughly removed, and it is generallybelieved that an efficient desizing is of crucial importance to thefollowing preparation processes: scouring and bleaching.

Scouring

According to the invention scouring may be carried out at conditionschosen to suit the process according to principles well known in theart. A scouring process employs sodium hydroxide (NaOH) or relatedcausticizing agents such as sodium carbonate, potassium hydroxide ormixtures thereof. Generally an alkali stable surfactant is added to theprocess to enhance solubilization of hydrophobic compounds and/orprevent their re-deposition back on the textile. The treatment isgenerally at a high temperature, i.e., 10° C.-100° C., preferably 40° C.to 60° C., employing strongly alkaline solutions, i.e., above pH 9,preferably 9-13, of the scouring agent. Due to the non-specific natureof chemical processes not only are the impurities but the, e.g.,cellulose itself is attacked, leading to damages in strength or otherdesirable textile properties. The softness of a cellulosic fabric is afunction of residual natural cotton waxes. The non-specific nature ofthe high temperature strongly alkaline scouring process cannotdiscriminate between the desirable natural cotton lubricants and themanufacturing introduced lubricants.

The scouring stage prepares the textile for the optimal response inbleaching. An inadequately scoured fabric will need a higher level ofbleach chemical in the subsequent bleaching stages.

Bleaching

According to the invention bleaching may be carried out using any knowprocess conditions in the art. In an embodiment the bleaching may becarried out at a temperature in the range of from about 30° C. to about100° C., more preferably from about 40° C. to about 90° C. The pH rangemay, dependent on the enzyme(s) applied, preferably be from about pH 5to about pH 11, more preferably from about pH 6 to about pH 8. Thereaction time may preferably be in the range of from about 15 minutes toabout 3 hours.

The term “bleaching” is here defined as a whitening of the textile. Thevalue of whiteness index (WI) is measured using a MacBeth Color Eyeequipped with Optiview 7000 software. The Whiteness index is calculatedfrom the following equation:WI=Y+800(x _(n) −x)+1700(y _(n) −y)where Y, x and y are chromaticity coordinates of the sample, and x_(n)and y_(n) are those of illuminant using the standard illuminant D65(imitating daylight).

METHODS OF THE INVENTION

As mentioned above, in the first aspect, the invention relates to qmethod of treating textiles, in particular fabrics, fibers, or yarnscomprising treating fabric, fiber, or yarn, in an aqueous medium, with acarbohydrate oxidase and/or a fatty acid oxidizing enzyme.

In a one embodiment the invention provides a method of treating fabrics,fibers, or yarns comprising treating fabric, fiber, or yarn in anaqueous medium with an effective amount of a carbohydrate oxidase havingactivity towards monosaccharides and at least one of di-saccharides andoligo-saccharides and a substrate for said carbohydrate oxidase.

Another embodiment of the invention provides a composition for use in amethod of treating fabrics, fibers, or yarns comprising a carbohydrateoxidase having activity towards monosaccharides and at least one ofdi-saccharides and oligo-saccharides and a substrate for saidcarbohydrate oxidase.

The treatment according to the present invention may be carried out atconditions chosen to suit the bleaching method according to principleswell known in the art. It will be understood that each of the reactionconditions, such as, e.g., concentration/dose of enzyme/substrate, pH,temperature, and time of treatment, may be varied, depending upon, e.g.,the source of the enzyme, the type of substrate, the method in which thetreatment is performed.

The method of the invention may further comprise the addition of one ormore chemicals capable of improving the enzyme-substrate interaction (inorder to improve the substrate's accessibility and/or dissolve reactionproducts), which chemicals may be added prior to, or simultaneously withthe enzymatic treatment. Such chemicals may in particular be wettingagents and dispersing agents etc., or mixtures thereof. Such chemicalsalso encompass peroxidase activators, e.g. silicate.

The enzymatic treatment according to the present invention preferably iscarried out as a wet process. An example of a suitable liquor:textileratio may be in the range of from about 20:1 to about 1:1, preferably inthe range of from about 15:1 to about 5:1.

The carbohydrate oxidase is generally added in an amount which iseffective to generate enough peroxide for providing the bleaching effectof the textile material. The enzyme(s) may preferably be dosed in anamount of from about 0.05 U/ml to about 10 U/ml of the total liquor,more preferably, from about 0.5 U/ml to about 5 U/ml, most preferably,from about 1 U/ml to about 3 U/ml.

The bleaching method can be carried out with the choice betweendifferent substrates either generated in situ with another enzyme orchemical system, from starch sizing and/or cellulosic fiber or added.

The amount of substrate employed in the method of the invention alsodepends on different parameters such as the enzyme applied. The amountof substrate is preferably from about 1 to about 200 mM of the totalliquor, more preferably, from about 3 to about 75 mM, even morepreferably, from about 10 to about 40 mM.

The enzymatic treatment is preferably carried out in a two step method,wherein the first step is a peroxide generating step in which theperoxide generating reaction is carried out. The second step is theactual bleaching step in which the textile material is contacted withthe generated peroxide.

In the first peroxide generating step, the fabric is incubated with thecarbohydrate oxidase and a suitable substrate, e.g., alpha-glucose, andoptionally other ingredients, such as buffer solution and surfactants,preferably at about 30° C. to about 50° C., more preferably, around 30°C., preferably at a pH in the range of about 5.5 to about 11, morepreferably, about 5.5 to about 9, and even more preferably at about 7preferably for 1 to 5 hours to generate peroxide. After incubation, thepH is preferably adjusted to a value above pH 7, such as, by adding analkaline solution, e.g. sodium hydroxide and the temperature ispreferably adjusted to a range of from about 75° C. to about 100° C.,more preferably, about 80° C. to about 95° C., and even more preferablyto around 90° C. The pH range is preferably in the range of about 10 toabout 13, more preferably above about 12. Bleaching is performed underthese conditions with the enzymatically produced peroxide, preferablyfor about 10 minutes to about 120 minutes, more preferably, about 30minutes to about 90 minutes, and even more preferably around 60 minutes.The fabric may also be added in the bleaching treatment after theperoxide generating step.

The method of the invention may optionally comprise a rinsing stepduring which the textile is rinsed in hot and cold water.

The materials may also be subject to additional processes. For example,for textile materials, the preparation may include the application offinishing techniques such as desizing and scouring, and other treatmentprocesses, such as imparting antimicrobial properties (e.g., usingquaternary ammonium salts), flame retardancy (e.g., by phosphorylationwith phosphoric acid or urea), increasing absorbency (by coating orlaminating with polyacrylic acid), providing an antistatic finish (e.g.,using amphoteric surfactants (N-oleyl-N,N-dimethylglycine)), providing asoil release finish (e.g., using NaOH), providing an antisoiling finish(e.g., using a fluorochemical agent), and providing an antipillingfinish (e.g., using NaOH, alcohol).

The method of the invention may be carried out in the presence ofconventional fabric, fiber, or yarn finishing agents, including wettingagents, polymeric agents, dispersing agents, etc.

A conventional wetting agent may be used to improve the contact betweenthe substrate and the enzyme used in the method. The wetting agent maybe a nonionic surfactant, e.g. an ethoxylated fatty alcohol. A preferredwetting agent is an ethoxylated and propoxylated fatty acid ester suchas Berol 087 (product of Akzo Nobel, Sweden).

Examples of suitable polymeris agents include proteins (e.g. bovineserum albumin, whey, casein or legume proteins), protein hydrolysates(e.g. whey, casein or soy protein hydrolysate), polypeptides,lignosulfonates, polysaccharides and derivatives thereof, polyethyleneglycol, polypropylene glycol, polyvinyl pyrrolidone, ethylene diaminecondensed with ethylene or propylene oxide, ethoxylated polyamines, orethoxylated amine polymers.

The dispersing agent may preferably be selected from nonionic, anionic,cationic, ampholytic or zwitterionic surfactants. More specifically, thedispersing agent may be selected from carboxymethylcellulose,hydroxypropylcellulose, alkyl aryl sulphonates, long-chain alcoholsulphates (primary and secondary alkyl sulphates), sulphonated olefins,sulphated monoglycerides, sulphated ethers, sulphosuccinates,sulphonated methyl ethers, alkane sulphonates, phosphate esters, alkylisothionates, acylsarcosides, alkyltaurides, fluorosurfactants, fattyalcohol and alkylphenol condensates, fatty acid condensates, condensatesof ethylene oxide with an amine, condensates of ethylene oxide with anamide, sucrose esters, sorbitan esters, alkyloamides, fatty amineoxides, ethoxylated monoamines, ethoxylated diamines, alcohol ethoxylateand mixtures thereof. A preferred dispersing agent is an alcoholethoxylate such as Berol 08 (product of Akzo Nobel, Sweden).

The bleaching processing may be performed using any machinery known inthe art.

The fabric may be further finished by one or more of the followingtreatments as are known in the art: dyeing, biopolishing, brightening,softening, and/or anti-wrinkling treatment(s).

In a second embodiment the invention relates to a method of treatingtextile, in particular fabrics, garments, or yarns, comprising a step oftreating the textile in an aqueous medium with one or more fatty acidoxidizing enzyme. The treatment may in embodiments of the invention becarried out in order to desized, scour and/or bleach textile as will beexplained further below.

Enzymes

The enzymatic method of the invention may be accomplished using anycarbohydrate oxidase enzyme which is capable of bleaching fabrics,fibers, and yarns in solution and/or a fatty acid oxidizing enzyme asdefined below.

Carbohydrate Oxidases

In the context of the present invention the term “carbohydrate oxidase”is intended to mean an enzyme selected from the group consisting ofenzymes classified under EC 1.1.3 (Enzyme Nomenclature;http://www.chem.qmw.ac.uk/iubmb/enzyme/).

Carbohydrate oxidases act on a very broad spectrum of substrates,including monosaccharides, such as glucose and xylose and di- andoligosaccharides, such as cellubiose and maltose.

Carbohydrate oxidase catalyses the following general reaction forperoxide generation at pH 5-8 and temperatures around 30-60° C.:R—CHO+O₂ R—COOH+H₂O₂

Based on the above mechanism, textile materials can be bleached byhydrogen peroxide, which is generated by carbohydrate oxidase duringoxidation of sugar substrates. The sugar substrate may be either addedor already present on the textile material as sizing materials.

Suitable substrates are mono-saccharides such as arabinose, xylose,α-glucose, β-gluconase, galactose, mannose, fructose, disaccharides suchas cellobiose, lactose, maltose, and oligo-saccharides such ascello-oligosaccharides and malto-oligosaccharides having a degree ofpolymerization of 3-6, particularly maltotriose, cellotriose,maltotetraose, and cellotetraose.

The enzyme has activity towards monosaccharides and at least one ofdi-saccharides and oligo-saccharides. The comparison between an enzymeof the present invention and an enzyme outside the scope of protectionmay be made at a substrate concentration of 1-200 mM and an enzymeconcentration of 0.05-10 U/ml by incubating the enzyme with thesubstrate at pH 5.5-11, and a temperature of 10-65° C. for 4 hours orless. Enzymes falling within the scope of the present invention showactivity towards at least one monosaccharide and at least one ofdi-saccharides and oligo-saccharides whereas enzymes falling outside thescope of the present invention show no activity towards at least onemonosaccharide and at least one of di-saccharide and oligosaccharide.

The carbohydrate oxidase may be derived from any origin, including,bacterial, fungal, yeast or mammalian origin.

The carbohydrate oxidase may be derived from a microbial source, such asa fungus, e.g. a filamentous fungus or yeast, in particular Ascomycotafungus, e.g. Euascomycetes, especially Pyrenomycetes such as Acremonium,in particular A. strictum.

The carbohydrate oxidase may further be derived from microorganisms ofXylariales, especially mitosporic Xylariales such as the genusMicrodochium, particularly the species M. nivale, more preferably M.nivale CBS 100236. Further microbial sources can be found in U.S. Pat.No. 6,165,761 which is hereby incorporated by reference.

The method of production of said enzyme is discloses in U.S. Pat. No.6,165,761 which is hereby incorporated by reference.

The Fatty Acid Oxidizing Enzyme

Any fatty acid oxidizing enzyme may be used according to the method ofthe invention. A fatty acid oxidizing enzyme is an enzyme whichhydrolyzes the substrate linoleic acid more efficiently than thesubstrate syringaldazine. “More efficiently” means with a higherreaction rate. This can be tested using the method described in Example9, and calculating the difference between (1) absorbancy increase perminute on the substrate linoleic acid (absorbancy at 234 nm), and (2)absorbancy increase per minute on the substrate syringaldazine(absorbancy at 530 nm), i.e., by calculating the Reaction RateDifference (RRD)=(d(A₂₃₄)/dt−d(A₅₃₀)/dt). If the RRD is above zero, theenzyme in question qualifies as a fatty acid oxidizing enzyme as definedherein. If the RRD is zero, or below zero the enzyme in question is nota fatty acid oxidizing enzyme.

In particular embodiments, the RRD is at least 0.05, 0.10, 0.15, 0.20,or at least 0.25 absorbancy units/minute.

In a particular embodiment of the method of Example 9, the enzymes arewell-defined. Still further, for the method of Example 9 the enzymedosage is adjusted so as to obtain a maximum absorbancy increase perminute at 234 nm, or at 530 nm. In particular embodiments, the maximumabsorbancy increase is within the range of 0.05-0.50; 0.07-0.4;0.08-0.3; 0.09-0.2; or 0.10-0.25 absorbancy units pr. min. The enzymedosage may for example be in the range of 0.01-20; 0.05-15; or 0.10-10mg enzyme protein per ml.

In the alternative, a “fatty acid oxidizing enzyme” may be defined as anenzyme capable of oxidizing unsaturated fatty acids more efficientlythan syringaldazine. The activity of the enzyme could be compared in astandard oximeter setup as described in Example 8 of the presentapplication at pH 6 and 30° C. including either syringaldazine orlinoleic acid as substrates.

In a particular embodiment, the fatty acid oxidizing enzyme is definedas an enzyme classified as EC 1.11.1.3, or as EC 1.13.11.-.EC1.13.11.-means any of the subclasses thereof, presently forty-nine: EC1.13.11.1-EC 1.13.11.49. EC 1.11.1.3 is designated fatty acidperoxidase, and EC 1.13.11.-is designated oxygenases acting on singledonors with incorporation of two atoms of oxygen.

In a further particular embodiment, the EC 1.13.11.-enzyme is classifiedas EC 1.13.11.12, EC 1.13.11.31, EC 1.13.11.33, EC 1.13.11.34, EC1.13.11.40, EC 1.13.11.44 or EC 1.13.11.45, designated lipoxygenase,arachidonate 12-lipoxygenase, arachidonate 15-lipoxygenase, arachidonate5-lipoxygenase, arachidonate 8-lipoxygenase, linoleate diol synthase,and linoleate 11-lipoxygenase, respectively.

Lipoxygenase

In a preferred embodiment the fatty acid oxidizing enzyme is alipoxygenase (LOX), classified as EC 1.13.11.12, which is an enzyme thatcatalyzes the oxygenation of polyunsaturated fatty acids, especiallycis,cis-1,4-dienes, e.g., linoleic acid and produces a hydroperoxide.But also other substrates may be oxidized, e.g. monounsaturated fattyacids.

Microbial lipoxygenases can be derived from, e.g., Saccharomycescerevisiae, Thermoactinomyces vulgaris, Fusarium oxysporum, Fusariumproliferatum, Thermomyces lanuginosus, Pyricularia oryzae, and strainsof Geotrichum. The preparation of a lipoxygenase derived fromGaeumannomyces graminis is described in Examples 3-4 of WO 02/20730. Theexpression in Aspergillus oryzae of a lipoxygenase derived fromMagnaporthe salvinii is described in Example 2 of WO 02/086114, and thisenzyme can be purified using standard methods, e.g., as described inExample 4 of WO 02/20730.

Lipoxygenase (LOX) may also be extracted from plant seeds, such assoybean, pea, chickpea, and kidney bean. Alternatively, lipoxygenase maybe obtained from mammalian cells, e.g., rabbit reticulocytes.

Lipoxygenase activity may be determined as described in the Materials&Methods section.

The enzymatic treatment according to the present invention preferably iscarried out as a wet process. An example of a suitable liquor textileratio may be in the range of from about 20:1 to about 1:5, preferably inthe range of from about 15:1 to about 1:2, especially about 1:1.Examples of effective amounts of lipoxygenase (LOX) are from 0.001 to400 U/ml treatment liquor, preferably from 0.01 to 100 U/ml treatmentliquor, more preferably 0.05 to 50 U/ml treatment liquor, and even morepreferably 0.1 to 20 U/ml treatment liquor. Further optimization of theamount of lipoxygenase can hereafter be obtained using standardprocedures known in the art

Substrate

In a preferred embodiment the method of the invention is carried out inthe presence of a substrate of the fatty acid oxidizing enzyme. In anembodiment the fatty acid oxidizing enzyme is applied together with asubstrate for the enzyme capable of enhancing the enzymatic effect.Examples of such substrates are hydrolyzed oils such as oils fromsoybeans (rich in linoleic acid) or tall oil. Fatty acid substrates maybe released from the added oil by lipolytic enzymes or produced duringthe Kraft pulping or sulphate cooking.

In particular embodiments the substrate is a compound with 1,4-pentadienstructure, e.g. with cis,cis-1,4-pentadien structure, i.e. compoundshaving at least one such element in its structural formula. Examples ofsuch substrates are unsaturated fatty acids, e.g. palmitoleic acid,oleic acid, linoleic acid, linolenic acid, and arachidonic acid, as wellas their salts and esters, e.g. methyl- and ethyl-esters.

In further particular embodiments the substrate is linoleic acid;linoleic acid methyl or ethyl ester, linolenic acid, or linolenic acidmethyl or ethyl ester.

To explore the effect of adding a substrate for the fatty acid oxidizingenzyme in question, the following method may be used: The spectrum of 10mM abietic acid (emulsified in 0.2% Tween 20) is recorded.Characteristic peaks are observed around 200 nm and around 250 nm. In afirst experiment, a fatty acid oxidizing enzyme is added to the abieticacid emulsion. In a second experiment, a substrate for the fatty acidoxidizing enzyme is also added. The enzyme is e.g. a lipoxygenasederived from M. salvinii as described above, and the substrate is e.g.linoleic acid. The degradation of abietic acid is followedspectrophotometrically, and the peaks around 200 nm and around 250 nmdecrease more rapidly when linoleic acid is added together with thelipoxygenase.

In particular embodiments of the above method, and of the method of theinvention, the substrate, e.g., linoleic acid, is added in an amount of5-10000 ppm (mg/l), or 10-9000, 10-8000, 25-7500, 30-7000, 50-6000,50-5000, 50-4000, 75-3000, 75-2500, 80-2000, 90-1500, 100-1000, 150-800,or 200-700 ppm. In Example 11, 333 ppm of linoleic acid was usedtogether with a fatty acid oxidizing enzyme.

In further particular embodiments of the above method, and of the methodof the invention, the fatty acid oxidizing enzyme is used in an amountof 0.005-50 ppm (mg/l), or 0.01-40, 0.02-30, 0.03-25, 0.04-20, 0.05-15,0.05-10, 0.05-5, 0.05-1, 0.05-0.8, 0.05-0.6, or 0.1-0.5 ppm. The amountof enzyme refers to mg of a well-defined enzyme preparation.

Additional Enzymes

The carbohydrate oxidase and/or fatty acid oxidizing enzyme may be addedto the textile as the only enzyme(s), or may be used in combination withone or more additional enzymes. The term “an additional enzyme” means atleast one additional enzyme, e.g. one, two, three, four, five, six,seven, eight, nine, ten or even more additional enzymes. The additionalenzyme may be an amylase or a lipase.

The fatty acid oxidizing enzyme used in accordance with the presentinvention may be applied together with an additional enzyme selectedfrom the group consisting of: a proteolytic enzyme, such as a protease alipolytic enzyme, a cellulolytic enzyme, such as a cellulase, ahemicellulase, an amylolytic enzyme, such as a amyloglucosidase,pectolytic enzyme, such as a pectinase, an oxidoreductase, e.g., aperoxidase, a laccase, a glucose oxidase, a pyranose oxidase, alipooxygenase, and the like or mixtures hereof. In case of bleaching thetextile an oxidase, such as carbohydrate oxidase; or a peroxidase mayadvantageously be present. In scouring processes of the invention apectolytic enzyme, preferably a pectate lyase, may be used. Lipolyticenzymes, such as preferably cutinases and lipases, may be present duringscouring. For desizing of textile, amylolytic enzymes, such asalpha-amylases, may be present.

The additional enzyme may be of any origin, including mammalian andplant, and preferably of microbial (bacterial, yeast or fungal) originand may be derived by techniques conventionally used in the art. Theterm “derived” means in this context that the enzyme may have beenisolated from an organism where it is present natively, i.e. theidentity of the amino acid sequence of the enzyme are identical to anative enzyme. The term “derived” also means that the enzymes may havebeen produced recombinantly in a host organism, the recombinant producedenzyme having either an identity identical to a native enzyme or havinga modified amino acid sequence, e.g. having one or more amino acidswhich are deleted, inserted and/or substituted, i.e., a recombinantlyproduced enzyme which is a mutant and/or a fragment of a native aminoacid sequence or an enzyme produced by nucleic acid shuffling processesknown in the art. Within the meaning of a native enzyme are includednatural variants. Furthermore, the term “derived” includes enzymesproduced synthetically by, e.g., peptide synthesis. The term “derived”also encompasses enzymes which have been modified e.g. by glycosylation,phosphorylation, or by other chemical modification, whether in vivo orin vitro. The term encompasses an enzyme that has been isolated from anorganism where it is present natively, or one in which it has beenexpressed recombinantly in the same type of organism or another, orenzymes produced synthetically by, e.g., peptide synthesis. With respectto recombinantly produced enzymes the term “derived” refers to theidentity of the enzyme and not the identity of the host organism inwhich it is produced recombinantly.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is derived. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be “substantially pure,” that is, free fromother components from the organism in which it is produced, that is, forexample, a host organism for recombinantly produced enzymes. Inpreferred embodiment, the enzymes are at least 75% (w/w) pure, morepreferably at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% pure. In anotherpreferred embodiment, the enzyme is 100% pure.

The enzyme may be in any form suited for the use in the treatmentprocess, such as e.g. in the form of a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g. as disclosed in U.S. Pat. No.4,106,991 and U.S. Pat. No. 4,661,452, and may optionally be coated bymethods known in the art. Liquid enzyme preparations may, for instance,be stabilized by adding stabilizers such as a sugar, a sugar alcohol oranother polyol, lactic acid or another organic acid according toestablished methods. Protected enzymes may be prepared according to themethod disclosed in EP 238,216.

Some non-limiting examples of additional enzymes are listed below. Theenzymes written in capitals are commercial enzymes available fromNovozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. The activityof any of those additional enzymes can be analyzed using any methodknown in the art for the enzyme in question, including the methodsmentioned in the references cited.

Proteolytic Enzymes

Any proteolytic enzymes suitable for use in alkaline solutions can beused. Preferred are proteases including those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically orgenetically modified mutants are included. The protease may be a serineprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus, e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describedIn WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. ofporcine or bovine origin) and the Fusarium protease described in WO89/06270. Other proteases are derived from Nocardiopsis, Aspergillus,Rhizopus, Bacillus alcalophilus, B. cereus, B. natto, B. vulgatus, B.mycoide, and subtilisins from Bacillus, especially proteases from thespecies Nocardiopsis sp. and Nocardiopsis dassonvillei such as thosedisclosed in WO 88103947, and mutants thereof, e.g. those disclosed inWO 91/00345 and EP 415296.

Preferred commercially available protease enzymes include those soldunder the trade names ALCALASE™, SAVINASE™, PRIMASE™, NEUTRASE™,DURAZYM™, and ESPERASE™ by Novozymes A/S (Denmark), those sold under thetradename MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™ andPURAFECT OXP™ by Genencor International, and those sold under thetradename OPTICLEAN™ and OPTIMASE™ by Solvay Enzymes. Protease enzymesmay be incorporated into the compositions in accordance with theinvention at a level of from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level of from 0.0001% to 1% ofenzyme protein by weight of the composition, more preferably at a levelof from 0.001% to 0.5% of enzyme protein by weight of the composition,even more preferably at a level of from 0.01% to 0.2% of enzyme proteinby weight of the composition.

Lipolytic Enzymes

In the context of this invention lipolytic enzymes are classified inE.C. 3.1.1 and include true lipases, esterases, phospholipases, andlyso-phospholipases. More specifically the lipolytic enzyme may be alipase as classified by EC 3.1.1.3, EC 3.1.1.23 and/or EC 3.1.1.26, anesterase as classified by EC 3.1.1.1, EC 3.1.1.2, EC 3.1.1.6, EC3.1.1.7, and/or EC 3.1.1.8, a phospholipase as classified by EC 3.1.1.4and/or EC 3.1.1.32, a lyso-phospholipase as classified by EC 3.1.1.5 anda cutinase as classified in EC 3.1.1.74.

The lipolytic enzyme preferably is of microbial origin, in particular ofbacterial, of fungal or of yeast origin.

In a particular embodiment, the lipolytic enzyme used may be derivedfrom a strain of Absidia, in particular Absidia blakesleena and Absidiacorymbifera, a strain of Achromobacter, in particular Achromobacteriophagus, a strain of Aeromonas, a strain of Alternaria, in particularAlternaria brassiciola, a strain of Aspergillus, in particularAspergillus niger and Aspergillus flavus, a strain of Achromobacter, inparticular Achromobacter iophagus, a strain of Aureobasidium, inparticular Aureobasidium pullulans, a strain of Bacillus, in particularBacillus pumilus, Bacillus strearothermophilus and Bacillus subtilis, astrain of Beauvera, a strain of Brochothrix, in particular Bruchothrixthermosohata, a strain of Candida, In particular Candida cylindracea(Candida rugosa), Candida paralipolytica, Candida tsukubaensis, Candidaauriculariae, Candida humicola, Cadida foliarum, Candida cylindracea(Cadida rugosa) and Candida antarctica, a strain of Chromobacter, inparticular Chromobacter viscosum, a strain of Coprinus, in particularCoprinus cinerius, a strain of Fusarium, in particular Fusariumoxysporum, Fusarium solani, Fusarium solani pisi, and Fusarium roseumculmorum, a strain of Geotricum, in particular Geotricum penicillatum, astrain of Hansenula, in particular Hansenula anomala, a strain ofHumicola, in particular Humicola brevispora, Humicula lanuginosa,Humicola brevis var. thermoidea, and Humicola insolens, a strain ofHyphozyma, a strain of Lactobacillus, in particular Lactobacilluscurvatus, a strain of Metarhizium, a strain of Mucor, a strain ofPaecilomyces, a strain of Penicillium, in particular Penicilliumcyclopium, Penicillium crustosum and Penicillium expansum, a strain ofPseudomonas in particular Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia),Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia,Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonasalcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes,Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonaswisconsinensis, a strain of Rhizoctonia, in particular Rhizoctoniasolani, a strain of Rhizomucor, in particular Rhizomucor miehei, astrain of Rhizopus, in particular Rhizopus japonicus, Rhizopusmicrosporus and Rhizopus nodosus, a strain of Rhodosporidium, inparticular Rhodosporidium toruloides, a strain of Rhodotorula, inparticular Rhodotorula glutinis, a strain of Sporobolomyces, inparticular Sporobolomyces shibatanus, a strain of Thermomyces, inparticular Thermomyces lanuginosus (formerly Humicola lanuginosa), astrain of Thiarosporella, in particular Thiarosporella phaseolina, astrain of Trichoderma, in particular Trichoderma harzianum andTrichoderma reesei, and/or a strain of Verticillium.

In a more preferred embodiment, the lipolytic enzyme used according tothe invention is derived from a strain of Aspergillus, a strain ofAchromobacter, a strain of Bacillus, a strain of Candida, a strain ofChromobacter, a strain of Fusarium, a strain of Humicola, a strain ofHyphozyma, a strain of Pseudomonas, a strain of Rhizomucor, a strain ofRhizopus, or a strain of Thermomyces.

In a more preferred embodiment, the lipolytic enzyme used according tothe invention is derived from a strain of Bacillus pumilus, a strain ofBacillus stearothermophilis a strain of Candida cylindracea, a strain ofCandida antarctica, in particular Candida antarctica Lipase B (obtainedas described in WO 88/02775), a strain of Humicola insolens, a strain ofHyphozyma, a strain of Pseudomonas cepacia, or a strain of Thermomyceslanuginosus.

In the context of this invention biopolyester hydrolytic enzyme includeesterases and poly-hydroxyalkanoate depolymerases, in particularpoly-3hydroxyalkanoate depolymerases. In fact an esterase is a lipolyticenzyme as well as a biopolyester hydrolytic enzyme.

In a more preferred embodiment, the esterase is a cutinase or asuberinase. Also in the context of this invention, a cutinase is anenzyme capable of degrading cutin, cf. e.g. Lin T S & Kolattukudy P E,J. Bacteriol. 1978 133 (2) 942-951, a suberinase is an enzyme capable ofdegrading suberin, cf. e.g., Kolattukudy P E; Science 1980 208 990-1000,Lin T S & Kolattukudy P E; Physiol. Plant Pathol. 1980 17 1-15, and TheBiochemistry of Plants, Academic Press, 1980 Vol. 4 624-634, and apoly-3-hydroxyalkanoate depolymerase is an enzyme capable of degradingpoly-3-hydroxyalkanoate, cf. e.g. Foster et al., FEMS Microbiol. Lett.1994 118 279-282. Cutinases, for instance, differs from classicallipases in that no measurable activation around the critical micelleconcentration (CMC) of the tributyrine substrate is observed. Also,cutinases are considered belonging to a class of serine esterases. Thecutinase may also be a cutinase derived from Humicola insolens disclosedin WO 96/13580. The cutinase may be a variant such as one or thevariants disclosed in WO 00/34450 and WO 01/92502 which is herebyincorporated by reference.

The biopolyester hydrolytic enzyme preferably is of microbial origin, inparticular of bacterial, of fungal or of yeast origin.

In a preferred embodiment, the biopolyester hydrolytic enzyme is derivedfrom a strain of Aspergillus, in particular Aspergillus oryzae, a strainof Alternaria, in particular Alternaria brassiciola, a strain ofFusarium, in particular Fusarium solani, Fusarium solani pisi, Fusariumroseum culmorum, or Fusarium roseum sambuciumi, a strain ofHelminthosporum, in particular Helminthosporum sativum, a strain ofHumicola, in particular Humicola insolens, a strain of Pseudomonas, inparticular Pseudomonas mendocina, or Pseudomonas putida, a strain ofRhizoctonia, in particular Rhizoctonia solani, a strain of Streptomyces,in particular Streptomyces scabies, or a strain of Ulocladium, inparticular Ulocladium consortiale. In a most preferred embodiment thebiopolyester hydrolytic enzyme is a cutinase derived from a strain ofHumicola insolens, in particular the strain Humicola insolens DSM 1800.

In another preferred embodiment, the poly-3-hydroxyalkanoatedepolymerase is derived from a strain of Alcaligenes, in particularAlcaligenes faecalis, a strain of Bacillus, in particular Bacillusmegaterium, a strain of Camomonas, in particular Camomonas testosteroni,a strain of Penicillium, in particular Penicillium funiculosum, a strainof Pseudomonas, in particular Pseudomonas fluorescens, Pseudomonaslemoignei and Pseudomonas oleovorans, or a strain of Rhodospirillum, inparticular Thodospirillum rubrum.

Specific examples of readily available commercial lipases includeLIPOLASE™ (WO 98/35026) LIPOLASE™ Ultra, LIPOZYME™, PALATASE™, NOVOZYM™435, LECITASE™ (all available from Novozymes A/S, Denmark).

Examples of other lipases are LUMAFAST™, Ps. mendocian lipase fromGenencor Int. Inc.; LIPOMAX™, Ps. pseudoalcaligenes lipase from GistBrocades/Genenoor mnt. Inc.; Fusarium solani lipase (cutinase) fromUnilever; Bacillus sp. lipase from Solvay enzymes. Other lipases areavailable from other companies.

Examples of cutinases are those derived from Humicola insolens (U.S.Pat. No. 5,827,719); from a strain of Fusadum, e.g. F. roseum culmorum,or particularly F. solani pisi (WO 90/09446; WO 94/14964, WO 94/03578).The cutinase may also be derived from a strain of Rhizoctonia, e.g. R.solani, or a strain of Alternaria, e.g. A. brassicicola (WO 94/03578),or variants thereof such as those described in WO 00/34450, or WO01/92502.

Pectolytic Enzymes

The term “pectolytic enzyme” or “pectinase” as denoted herein, isintended to include any pectinase enzyme defined according to the artwhere pectinases are a group of enzymes that hydrolyse glycosidiclinkages of pectic substances mainly poly-1,4-a-D-galacturonide and itsderivatives(see reference Sakai et al., Pectin, pectinase andpropectinase: production, properties and applications, pp 213-294 in:Advances in Applied Microbiology vol: 39,1993) which enzyme isunderstood to include a mature protein or a precursor form thereof or afunctional fragment thereof which essentially has the activity of thefull-length enzyme. Furthermore, the term “pectolytic” enzyme isintended to include homologues or analogues of such enzymes.

Preferably a pectolytic enzyme useful in the method of the invention isa pectinase enzyme which catalyzes the random cleavage ofalpha-1,4-glycosidic linkages in pectic acid also calledpolygalacturonic acid by transelimination such as the enzyme classpolygalacturonate lyase (EC 4.2.2.2) (PGL) also known aspoly(1,4-a-D-galacturonide)lyase also known as pectate lyase. Alsopreferred is a pectinase enzyme which catalyzes the random hydrolysis ofalpha-1,4-glycosidic linkages in pectic acid such as the enzyme classpolygalacturonase (EC 3.2.1.15) (PG) also known as endo-PG. Alsopreferred is a pectinase enzyme such as polymethylgalcturonate lyase (EC4.2.2.10) (PMGL), also known as Endo-PMGL, also known aspoly(methyoxygalacturonide)lyase also known as pectin lyase whichcatalyzes the random cleavage of alpha-1,4-glycosidic linkages ofpectin. Other preferred pectinases are galactanases (EC 3.2.1.89),arabinanases (EC 3.2.1.99), pectin esterases (EC 3.1.1.1.1), andmannanases (EC 3.2.1.78).

The enzyme Is preferably derived from a microorganism, preferably from abacterium, an archea or a fungus, especially from a bacterium such as abacterium belonging to Bacillus, preferably to an alkalophilic Bacillusstrain which may be selected from the group consisting of the speciesBacillus licheniformis and highly related Bacillus species in which allspecies are at least 90% homologous to Bacillus licheniformis based onaligned 16S rDNA sequences. Specific examples of such species are thespecies Bacillus licheniformis, Bacillus alcalophilus, Bacilluspseudoalcalophilus, and Bacillus clarkii. A specific and highlypreferred example is the species Bacillus licheniformis, ATCC 14580.Other useful pectate lyases are derivable from the species Bacillusagaradhaerens, especially from the strain deposited as NCIMB 40482; andfrom the species Aspergillus aculeatus, especially the strain and theenzyme disclosed in WO 94/14952 and WO 94/21786 which are herebyincorporated by reference in their entirety; and from the speciesBacillus subtilis, Bacillus stearothermophilus, Bacillus pumilus,Bacillus cohnii, Bacillus pseudoalcalophilus, Erwinia sp. 9482,especially the strain FERM BP-5994, and Paenibacillus polymyxa.

The pectolytic enzyme may be a component occurring in an enzyme systemproduced by a given microorganism, such an enzyme system mostlycomprising several different pectolytic enzyme components includingthose identified above.

Alternatively, the pectolytic enzyme may be a single component, i.e. acomponent essentially free of other pectinase enzymes which may occur inan enzyme system produced by a given microorganism, the single componenttypically being a recombinant component, i.e. produced by cloning of aDNA sequence encoding the single component and subsequent celltransformed with the DNA sequence and expressed in a host. Such usefulrecombinant enzymes, especially pectate lyases, pectin lyases andpolygalacturonases are described in detail in e.g. WO 99/27083 and WO99/27084 (from Novozymes A/S) which are hereby incorporated by referencein their entirety including the sequence listings. The host ispreferably a heterologous host, but the host may under certainconditions also be the homologous host.

In a preferred embodiment the pectate lyase used according to theinvention is derived from the genus Bacillus, preferably the speciesBacillus licheniformis.

The pectate lyase is normally incorporated in the composition at a levelof from 0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

Commercially available products include BIOPREP™ from Novozymes A/S,Denmark.

Amylolytic Enzymes

Preferred amylolytic enzymes are amylases. Any amylase (alpha and/orbeta) suitable for use in alkaline solutions can be used. Suitableamylases include those of bacterial or fungal origin. Chemically orgenetically modified mutants are included. Amylases include, forexample, alpha-amylases obtained from a special strain of B.licheniformis, described in more detail in GB 1,296,839. Commerciallyavailable amylases are DURAMYL™, NATALASE™, TERMAMYL™, STAINZYME™,AQUAZYM™, and AQUAZYM™ Ultra, FUNGAMYL™ and BAN™ (available fromNovozymes A/S) and RAPIDASE™ and MAXAMYL P™ (available from GenencorInt., USA).

The amylase(s) is(are) normally incorporated in the composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Cellulolytic Enzyme

In the present context, the term “cellulase or “cellulolytic enzyme”refers to an enzyme which catalyzes the degradation of cellulose toglucose, cellobiose, triose and other cellooligosaccharides. Celluloseis a polymer of glucose linked by beta-1,4-glucosidic bonds. Cellulosechains form numerous intra- and intermolecular hydrogen bonds, whichresult in the formation of insoluble cellulose microfibrils. Microbialhydrolysis of cellulose to glucose involves the following three majorclasses of cellulases: endo-1,4-beta-glucanases (EC 3.2.1.4), whichcleave beta-1,4-glucosidic links randomly throughout cellulosemolecules; cellobiohydrolases (EC 3.2.1.91)(exoglucanases), which digestcellulose from the nonreducing end; and beta-glucosidases (EC 3.2.1.21),which hydrolyse cellobiose and low-molecular-mass cellodextrins torelease glucose. Most cellulases consist of a cellulose-binding domain(CBD) and a catalytic domain (CAD) separated by a linker rich in prolineand hydroxy amino acid residues. In the specification and claims, theterm “endoglucanase” is intended to denote enzymes with cellulolyticactivity, especially endo-1,4-beta-glucanase activity, which areclassified in EC 3.2.1.4 according to the Enzyme Nomenclature (1992) andare capable of catalysing (endo)hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, lichenin and cereal beta-D-glucans including1,4-linkages in beta-D-glucans also containing 1,3-linkages. Anycellulase suitable for use in alkaline solutions can be used. Suitablecellulases include those of bacterial or fungal origin. Chemically orgenetically modified mutants are included. Suitable cellulases aredisclosed in U.S. Pat. No. 4,435,307, which discloses fungal cellulasesproduced from Humicola insolens. Especially suitable cellulases are thecellulases having colour care benefits. Examples of such cellulases arecellulases described in European patent application No. 0 495 257, WO91/17243 and WO 96/29397.

Commercially available cellulases include CELLUZYME™ and DENIMAX™produced by a strain of Humicola insolens (Novozymes A/S), andKAC500(B)™ (Kao Corporation).

Cellulases are normally incorporated in the composition at a level offrom 0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

Peroxidases/Oxidases

Peroxidase enzymes are used in combination with hydrogen peroxide or asource thereof (e.g. a percarbonate, perborate or persulfate). Oxidaseenzymes are used in combination with oxygen. Both types of enzymes areused for “solution bleaching”, i.e. to prevent transfer of a textile dyefrom a dyed fabric to another fabric when said fabrics are washedtogether in a wash liquor, preferably together with an enhancing agentas described in e.g. WO 94/12621 and WO 95/01426. Suitableperoxidases/oxidases include those of plant, bacterial or fungal origin.Chemically or genetically modified mutants are included.

Peroxidase and/or oxidase enzymes are normally incorporated in thecomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a protease, an amylase, a lipase and/or acellulase.

The enzyme of the invention, or any other enzyme incorporated in thecomposition, is normally incorporated in the composition at a level from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level from 0.0001% to 1% of enzyme protein by weight ofthe composition, more preferably at a level from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel from 0.01% to 0.2% of enzyme protein by weight of the composition.

Bleach Activator

Any suitable bleach activator may be employed in the present invention.The bleach activators preferred for use in accordance with theinvention, include, for example, compounds of the following classes ofsubstances: Polyacylated sugars or sugar derivatives with C sub1-10-acyl radicals, preferably acetyl, propionyl, octanoyl, nonanoyl orbenzoyl radicals, particularly preferably acetyl radicals, can be usedas bleach activators. Sugars or sugar derivatives which can be used aremono- or disaccharides and their reduced or oxidized derivatives,preferably glucose, mannose, fructose, sucrose, xylose or lactose.Particularly suitable bleach activators of this class of substances are,for example, pentaacetylglucose, xylose tetraacetate,1-benzoyl-2,3,4,6-tetraacetylglucose and1-octanoyl-2,3,4,6-tetraacetylglucose.

Another class of substances which are preferred for use as bleachactivators in the present invention comprises acyloxybenzenesulfonicacids and their alkali metal and alkaline earth metal salts, such as Csub 1-14-acyl radicals. Acetyl, propionyl, octanoyl, nonanoyl andbenzoyl radicals are preferred, especially acetyl radicals and nonanoylradicals. Particularly suitable bleach activators in this class ofsubstances are acetyloxybenzenesulfonic acid andbenzoyloxybenzenesulfonic acid. They are preferably employed in the formof their sodium salts.

Other bleach activators for use in the present invention include MMA andOCL, alone or in combination with each other or with TAED; O-acyloximeesters, such as acetone O-acetyloxime, acetone O-benzoyloxime,bis(propylimino)carbonate, bis(cyclohexylimino)carbonate as a bleachactivator. Acylated oximes which can be used as a bleach activatoraccording to the invention are described, for example, in EP-A-0 028432. Oxime esters which can be used as a bleach activator according tothe invention are described, for example in EP-A-0 267 046.

Additional preferred bleach activators include N-acylcaprolactams, suchas N-acetylcaprolactam, N-benzoylcaprolactam, N-octanoylcaprolactam andcarbonylbiscaprolactam; N,N-diacylated and N,N,N′,N′-tetraacylatedamines, such as N,N,N′,N′-tetraacetylmethylenediamine and-ethylenediamine (TAED), N,N-diacetylaniline, N,N-diacetyl-p-toluidineor 1,3diacylated hydantoins such as 1,3-diacetyl-5,5-dimethylhydantoin;N-alkyl-N-sulfonylcarboxamides, such as N-methyl-N-mesylacetamide orN-methyl-N-mesylbenzamide; N-acylated cyclic hydrazides, acylatedtriazoles or urazoles, such as monoacetylated maleic hydrazide;O,N,N-trisubstituted hydroxylamines, such asO-benzoyl-N,N-succinyl-hydroxylamine,O-acetyl-N,N,N-succinylhydroxylamine or O,N,N-triacetylhydroxylamine;N,N′-diacylsulfamides, such as N,N′-dimethyl-N,N′-diacetylsulfamide orN,N′-diethyl-N,N′-diproplonylsulfamide; triacylcyanurates, such astriacetylcyanurate or tribenzoylcyanurate; carboxylic anhydrides, suchas benzoic anhydride, m-chlorobenzoic anhydride or phthalic anhydride;1,3-diacyl-4,5-diacyloxyimidazolines, such as1,3-diacetyl-4,5-diacetoxyimidazoline; tetraacetylglycoluril andtetrapropionylglycoluril; diacylated 2,5-diketopiperazines, such as1,4-diacetyl-2,5-diketopiperazine; acylation products of propylenediureaand 2,2-dimethylpropylenediurea, such as tetraacetylpropylenediurea;alpha.-acyloxypolyacylmalonamides, such as.alpha.-acetoxy-N,N′-diacetylmalonamide;diacyldioxohexahydro-1,3,5-triazines, such as1,5-diacetyl-2,4dioxohexahydro-1,3,5-triazine; 2-alkyl- or2-aryl-(4H)-3,1-benzoxazin-4-ones as described, for example, in EP-B1-0332 294 and EP-B 0 502 013, and 2-phenyl-(4H)-3,1-benzoxazin-4-one and2-methyl-(4H)-3,1-benzoxazin-4-one, cationic nitrites, as described, forexample, in EP 303 520 and EP 458 396 A1, such as, methosulfates ortosylates of trimethylammonioacetonitrile,N,N-dimethyl-N-octylammonioacetonitrile,2-(trimethylammonio)propionitrile,2-(trimethylammonio)-2-methylpropionitrile. Also suitable are themethosulfates of N-methylpiperazinio-N,N′-diacetonitrile andN-methylmorpholinioacetonitrile (MMA).

Additional bleach activators for use in the present invention includepercarbamic acids or diacyl percarbamates and precursors thereof, asdisclosed, e.g., in WO 02/16538 hereby incorporated by reference.

Bleach activators are typically added in an amount from about 0.1 to 30g/l, more preferably 0.5 to 10 g/l.

Bleach Stabilizer

In another preferred embodiment of the present invention, the bleachingsystem additionally contains one or more bleach stabilizers. The bleachstabilizers comprise additives able to adsorb, bind or complex traces ofheavy metals. Examples of additives which can be used according to theinvention with a bleach-stabilizing action are polyanionic compounds,such as polyphosphates, polycarboxylates, polyhydroxypolycarboxylates,soluble silicates as completely or partially neutralized alkali metal oralkaline earth metal salts, in particular as neutral Na or Mg salts,which are relatively weak bleach stabilizers. Examples of strong bleachstabilizers which can be used according to the invention are complexingagents such as ethylenediaminetetraacetate (EDTA),diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA),methyl-glycinediacetic acid (MGDA), .beta.-alaninediacetic acid (ADA),ethylenediamine-N,N′-disuccinate (EDDS) and phosphonates such asethylenediaminetetramethylenephosphonate,diethylenetriaminepentamethylenephosphonate (DTMPA) orhydroxyethylidene-1,1-diphosphonic add in the form of the acids or aspartially or completely neutralized alkali metal salts.

The bleach stabilizer is typically added to the treating composition inan amount from about 0.1 to about 5/g liter of the composition, morepreferably from about 0.5 to about 2 g/l, and most preferably about 1g/l.

Adjuvants

The method of the invention may be carried out in the presence ofconventional textile adjuvants, including fabric, fiber, or yarnfinishing agents, including wetting agents, such as certain surfactants;polymeric agents; dispersing agents, etc.

Wetting Agents

A conventional wetting agent may be used to improve the contact betweenthe substrate and the enzyme used in the method. The wetting agent maybe a nonionic surfactant, e.g. an ethoxylated fatty alcohol. A preferredwetting agent is an ethoxylated and propoxylated fatty acid ester suchas Berol 087 (product of Akzo Nobel, Sweden). In an embodiment themethod of the invention is carried out in the presence of a surfactant.Preferred surfactants are nonionic, non-linear surfactants. The term“nonionic” is well defined in the literature and generally refers tosurfactants that do not possess ionizable functional groups. In thecontext of the present invention, the term “non-linear” is defined as asurfactant whose hydrophobic portion of the molecular structure is of abranched origin and possesses chain branching. Chain branching isdefined in the context of the present invention as a molecular structurepossessing one or more carbon atoms directly bonded to more than twocarbon atoms or whose hydrophobic portion is derived from a secondary ortertiary alcohol. Polyethylene, polypropylene, and polybutylene oxidecondensates of alkyl phenols are suitable for use as the nonionic,non-linear surfactant of the surfactant systems of the presentinvention, with the polyethylene oxide condensates being preferred.These compounds include the condensation products of alkyl phenolshaving an alkyl group containing from about 6 to about 14 carbon atoms,preferably from about 8 to about 14 carbon atoms, in either a straightchain or branched-chain configuration. In a preferred embodiment, theethylene oxide is present in an amount equal to from about 2 to about 25moles, more preferably from about 3 to about 15 moles, of ethylene oxideper mole of alkyl phenol. Commercially available nonionic, nonlinearsurfactants of this type include Igepal™ CO-630, marketed by the GAFCorporation, Triton™ X-45, X-114, X-100 and X-102, and Terginol NP,preferably Terginol NP9 all marketed by DOW/Union Carbide. Thesesurfactants are commonly referred to as alkylphenol alkoxylates (e.g.,alkyl phenol ethoxylates).

The condensation products of secondary aliphatic alcohols with about 1to about 25 moles of ethylene oxide are suitable for use as the nonionicsurfactant of the nonionic surfactant systems of the present invention.The alkyl chain of the aliphatic alcohol generally contains from about 8to about 22 carbon atoms. Preferred are the condensation products ofalcohols having an alkyl group containing from about 8 to about 20carbon atoms, more preferably from about 10 to about 18 carbon atoms,with from about 2 to about 15 moles of ethylene oxide per mole ofalcohol, preferably about 5 to about 15 moles of ethylene oxide and mostpreferably from about 7 to about 13 moles of ethylene oxide per mole ofalcohol. Examples of commercially available nonionic surfactants of thistype include Terginol™ 15-S-9 (the condensation product of C₁₁-C₁₅secondary alcohol with 9 moles ethylene oxide), Terginol™ 15-S-12 andSoftanol 90. Preferred range of HLB in these products is from 8-15 andmost preferred from 10-14. The condensation products of secondaryaliphatic alcohols with about 1 to about 25 moles of ethylene oxide aresuitable for use as the nonionic surfactant of the nonionic surfactantsystems of the present invention. The alkyl chain of the aliphaticalcohol generally contains from about 8 to about 22 carbon atoms.Preferred are the condensation products of alcohols having an alkylgroup containing from about 8 to about 20 carbon atoms, more preferablyfrom about 10 to about 18 carbon atoms, with from about 2 to about 15moles of ethylene oxide per mole of alcohol, preferably about 5 to about15 moles of ethylene oxide and most preferably from about 7 to about 13moles-of ethylene oxide per mole of alcohol. Examples of commerciallyavailable nonionic surfactants of this type include Terginol™ 15-S-9(the condensation product of C₁₁-C₁₅ secondary alcohol with 9 molesethylene oxide), Terginol™ 15-S-12 and Softanol 90. Preferred range ofHLB in these products is from 8-15 and most preferred from 10-14.

Also useful as the nonionic surfactant of the surfactant systems of thepresent invention are the condensation products of styrenated phenolicswith ethylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 9 to about 15 moles, of ethylene oxide per mole ofstyrenated phenol. Examples of commercially available styrenated phenolsof this type are Ethox 2622, Ethox 2659 and Ethox 2938.

The condensation products of branched aliphatic alcohols such astridecylalcohol with about 1 to about 25 moles of ethylene oxide aresuitable for use as the nonionic surfactant of the nonionic surfactantsystems of the present invention. Commercially available examples ofthis surfactant class are Novell II TDA6.6, Novell II TDA-7, Novell IITDA8.5, Novell II TDA-9, Novell II TDA-9.5 and Novell II TDA-11.

Polymeric Agents

Examples of suitable polymeric agents Include proteins (e.g. bovineserum albumin, whey, casein or legume proteins), protein hydrolysates(e.g. whey, casein or soy protein hydrolysate), polypeptides,lignosulfonates, polysaccharides and derivatives thereof, polyethyleneglycol, polypropylene glycol, polyvinyl pyrrolidone, ethylene diaminecondensed with ethylene or propylene oxide, ethoxylated polyamines, orethoxylated amine polymers.

Dispersing Agents

The dispersing agent may preferably be selected from nonionic, anionic,cationic, ampholytic or zwitterionic surfactants. More specifically, thedispersing agent may be selected from carboxymethylcellulose,hydroxypropylcellulose, alkyl aryl sulphonates, long-chain alcoholsulphates (primary and secondary alkyl sulphates), sulphonated olefins,sulphated monoglycerides, sulphated ethers, sulphosuccinates,sulphonated methyl ethers, alkane sulphonates, phosphate esters, alkylisothionates, acylsarcosides, alkyltaurides, fluorosurfactants, fattyalcohol and alkylphenol condensates, fatty acid condensates, condensatesof ethylene oxide with an amine, condensates of ethylene oxide with anamide, sucrose esters, sorbitan esters, alkyloamides, fatty amineoxides, ethoxylated monoamines, ethoxylated diamines, alcohol ethoxylateand mixtures thereof. A preferred. dispersing agent is an alcoholethoxylate such as Berol 08 (product of Akzo Nobel, Sweden).

The textile may be further finished by one or more of the followingtreatments as are known in the art: dyeing, biopolishing, brightening,softening, and/or anti-wrinkling treatment(s).

Composition

In a final aspect the invention relates to a composition comprising afatty acid oxidizing enzyme and in addition thereto at least oneadjuvant.

In a preferred embodiment the adjuvant is selected from the groupconsisting of wetting agent, polymeric agent, and dispersing agent.

The fatty acid oxidizing enzyme may be any of the above mentioned.Preferred fatty acid oxidizing enzymes are lipoxygenases, especially theabove mentioned derived from the genus Magnaporthe, especially a strainof Magnaporthe salvinii.

The composition of the invention may in a preferred embodiment furthercomprising an enzyme selected from the group consisting of: aproteolytic enzyme, a lipolyuc enzyme, a cellulolytic enzyme, anamylolytic enzyme, a pectolytic enzyme, an oxidase enzyme, or aperoxidase enzyme, or mixtures hereof. Preferred additional enzymes arecutinases, amylases and pectate lyases.

The invention is further illustrated in the following examples, whichare not intended to be in any way limiting to the scope of the inventionas claimed.

Materials and Methods

Materials:

Enzymes

Microdochium nivale carbohydrate oxidase, expressed and purified fromFusarium venenatum (U.S. Pat. No. 6,165,761).

Fatty acid oxidizing enzyme: Lipoxygenase from Magnaporthe salvinii wascloned and expressed in Aspergillus oryzae as described in Example 2 ofWO 02/086114.

Pectate lyase: Bioprep 3000L (batch KND00007, 3000 APSU/g) availablefrom Novozymes A/S, Denmark.

Cutinase: (2002-00081, 17.2 KLU/g) is disclosed in WO 01/92502 and isderived from the wild-type cutinase of Humicola insolens DSM 1800comprising the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E,R51P, A88H, N91H, A130V, E179Q and R189V and is available from NovozymesA/S, Denmark.

AQUAZYM™ ULTRA is an alpha-amylase available from Novozymes A/S,Denmark.

Na₂HPO₄ 7H₂O (F. W. 268.07, US-0001-10) was purchased from FisherScientific.

Na₂B₄O₇ 10H₂O (F.W. 381.37, US-0064-11) was purchased from Aldrich.

Na₂CO₃ H₂O was purchased from Aldrich.

Kieralon Jet B is a mixture of nonionic surfactants purchased from BASF.

Linoleic acid (99%, batch 71k2050) was purchased from SIGMA (USA).

Linoleic acid (L-1376, lot 61K1147) was purchased from SIGMA (USA).

Linolenic acid (L-2376, lot 072K1228) was purchased from SIGMA (USA).

Medium and Substrates

Surfactant Kierlon Jet B available from BASF.

Na-phosphate buffer pH 7.0. Prepared by mixing 20 mM NaH₂PO₄ and 1 NNaOH.

D-arabinose: Aldrich

D-xylose: Aldrich

D-alpha-glucose: Sigma-Aldrich (Cat. 15,896-8)

D-beta-glucose: SIGMA (G-5250)

D-galactose: Sigma (G-065)

D-fructose: Sigma (F2543)

D-mannose: Fisher Scientific (M-12175767)

D-cellobiose: Sigma-Aldrich(C-7252)

D-maltose: Sigma (M9171)

D-beta-lactose: Sigma (L-3750)

Maltotriose: Sigma (M-8378)

Dextrin: Sigma (75%, type III, from corn)

Sodium hydroxide available from Fisher Scientific Co.

Equipment

MacBeth Color Eye equipped with Optiview 7000 software.

Labomat (Mathis)

Methods

Whiteness index (WI) was calculated from the following equation:WI=Y+800(x _(n) −x)+1700(y _(n) −y)where Y, x and y are chromaticity coordinates of the sample, and x_(n)and y_(n) are those of illuminant using the standard illuminant D65. Thewater absorbency of the swatches was determined according to AATCCmethod 79 (Technical Manual of the American Association of TextileChemists and Colorists).Carbohydrate Oxidase Activity (COXU)

A Carbohydrate Oxidase unit (COXU is defined as the amount of enzymethat oxidizes one micromol lactose per minute under the followingconditions: Buffer 100 mm phosphate - 100 mm citrate pH 6.0 carbohydrateoxidase 0.2-1 micro g enzyme/ml lactose 4.3 mm 4-aminoantipyrine {AA}1.7 mm n-ethyl-n-sulfopropyl-m- 4.3 mm toluidine {TOPS} peroxidase,sigma 2.1 U/mL temperature 37° C. time 4.17 minutes wavelength 550 nm

Here is one COXU defined as one mg of pure carbohydrate oxidaseenzyme—relative to an enzyme standard. carbohydrate oxidase acts in thepresence of o₂ on lactose to form lactobionic acid and H₂O₂. The formedH₂O₂ activates in the presence of peroxidase the oxidative condensationof 4-aminoantipyrine {AA} and n-ethyl-n-sulfopropyl-m-toluidine {TOPS},to form a purple product which can be quantified by its absorbance at550 nm. When all components but carbohydrate oxidase are in surplus, therate of the rising absorbance is proportional to the COXU, carbohydrateoxidase activity present. The reaction proceeds automatically in theCobas Fara centrifugal analyzer.

Lipoxygenase Activity (LOX Units)

The lipoxygenase activity was measured according to Novozymes; StandardMethod 2001-21910-03 hereby incorporated by reference and available fromNovozyme A/S, Denmark, on request One LOX unit causes an increase inA₂₃₄ of 0.001 per minute at ph 9.0 at 30° C., when linoleic acid is usedas substrate. Reaction volume=1.0 ml (1 cm light path).

Cutinase Activity (LU)

The cutinase activity is determined as lipolytic activity determinedusing tributyrine as substrate. This method was based on the hydrolysisof tributyrin by the enzyme, and the alkali consumption is registered asa function of time. One Lipase Unit (LU) is defined as the amount ofenzyme which, under standard conditions (i.e. at 30.0° C.; pH 7.0; withGum Arabic as emulsifier and tributyrine as substrate) liberates 1micromol titrable butyric acid per minute. A folder AF 95/5 describingthis analytical method in more detail is available upon request toNovozymes A/S, Denmark, which folder is hereby included by reference.

Determination of Pectate Lyase Activity

The Viscosity Assay APSU

APSU units: The APSU unit assay is a viscosity measurement using thesubstrate polygalacturonic acid with no added calcium.

The substrate 5% polygalacturonic acid sodium salt (Sigma P-1879) issolubilised in 0.1 M Glycin buffer pH 10. The 4 ml substrate ispreincubated for 5 min at 40° C. The enzyme is added (in a volume of 250microliters) and mixed for 10 sec on a mixer at maximum speed, it isthen incubated for 20 min at 40° C. For a standard curve doubledetermination of a dilution of enzyme concentration in the range of 5APSU/ml to above 100 APSU/ml with minimum of 4 concentrations between 10and 60 APSU per ml.

The viscosity is measured using a MIVI 600 from the company Sofraser,45700 Villemandeur, France. The viscosity is measured as mV after 10sec.

For calculation of APSU units a enzyme standard dilution as describedabove was used for obtaining a standard curve. The GrafPad Prismprogram, using a non linear fit with a one phase exponential decay witha plateau, was used for calculations. The plateau plus span is the mVobtained without enzyme. The plateau is the mV of more than 100 APSU andthe half reduction of viscosity in both examples was found to be 12 APSUunits with a standard error of 1.5 APSU.

The Lyase Assay (at 235 nm)

For determination of the β-elimination an assay measuring the increasein absorbance at 235 nm was carried out using the substrate 0.1%polygalacturonic acid sodium salt (Sigma P-1879) solubilized in 0.1 MGlycin buffer pH 10. For calculation of the catalytic rate an increaseof 5.2 Absorbency at 235 units per min corresponds to formation of 1micro-mol of unsaturated product (Nasuna and Starr (1966) J. Biol. Chem.Vol 241 page 5298-5306; and Bartling, Wegener and Olsen (1995)Microbiology Vol 141 page 873-881).

Steady state condition using a 0.5 ml cuvette with a 1 cm light; path ona HP diode array spectrophotometer in a temperature controlled cuvetteholder with continuous measurement of the absorbency at 235 nm. Forsteady state a linear increase for at least 200 sec was used forcalculation of the rate. It was used for converted to formation μmol permin product.

Determination of Cellulase Activity (ECU)

The cellulolytic activity may be determined in endo-cellulase units(ECU) by measuring the ability of the enzyme to reduce the viscosity ofa solution of carboxy methyl cellulose (CMC).

The ECU assay quantifies the amount of catalytic activity present in thesample by measuring the ability of the sample to reduce the viscosity ofa solution of carboxy-methylcellulose (CMC). The assay is carried out ina vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France) at 40°C.; pH 7.5; 0.1 M phosphate buffer; time 30 min; using a relative enzymestandard for reducing the viscosity of the CMC substrate(Hercules 7LFD), enzyme concentration approx. 0.15 ECU/ml. The arch standard isdefined to 8200 ECU/g.

One ECU is amount of enzyme that reduces the viscosity to one half underthese conditions.

EXAMPLES Example 1 Effect of Glucose, Carbohydrate Oxidase and NaOH inCotton Bleaching

Peroxide generation and bleaching was conducted in Labomat (Mathis).Typically, about 140 ml sodium phosphate buffer, 0.5 g/l Kierlon Jet B,alpha-glucose and carbohydrate oxidase were added to a 1 liter beaker,containing two fabric swatches about 14 g total weight of cotton knit(Ramseur). All beakers were incubated at 40° C. for 4 hours to generateperoxide. Sodium hydroxide was added and the beaker temperature wasraised to 95° C. After 60 minutes incubation, all beakers were cooled to80° C. Cotton swatches were taken out and the liquor pH in the beakerwas measured. Cotton swatches were rinsed in hot (50° C.) and cold waterfor 10 minutes prior to air drying. All beakers were constantly rotatedat 50 rpm.

After cotton swatches were equilibrated in a constant temperature andhumidity (70° F., 65% RH) for at least 24 hours, the value of whitenessindex (WI) was measured using MacBeth Color Eye.

Results are shown in Table 1 below. Cotton swatches in four beakers havedifferent whiteness index. Glucose alone reduces cotton fabricwhiteness. Carbohydrate oxidase and sodium hydroxide improve cottonwhiteness. TABLE 1 Peroxide generation Sam- Carbohydrate Bleaching pleOxidase Glucose NaOH Final CIE Absorbency # (U/ml) (g/l) (g/l) pH WI(second) S1 0 0 0 7.0 26.46 <1 S2 0 5 0 6.9 24.86 N/A* S3 0 5 0.5 7.426.22 <1 S4 1.5 5 0.5 6.6 33.20 <1*N/A: not measured.

Example 2 Effect of Peroxide Generation Time, Glucose and CarbohydrateOxidase on Fabric Properties

All materials and chemicals were essentially the same as in Example 1.The peroxide generation and bleach experimental were the same as inExample 1 except that the time, the amount of Carbohydrate Oxidase andglucose varied during peroxide generation, the concentration of sodiumhydroxide was kept at 3 g/l. The value of whiteness index and absorbencyof cotton fabric were measured using the same methods in Example 1.After bleaching, liquor pH was measured and liquor color was alsoobserved and recorded.

Table 2 shows the value of whiteness index and absorbency results.Statistic analysis indicated that Carbohydrate Oxidase and time arestatistically significant. Increasing carbohydrate oxidase (from 0.5U/ml to 1.5 U/ml) enhanced whiteness by 6 units. Higher pH in thebleaching step appeared promoting the bleaching performance. TABLE 2Impact of time and Carbohydrate Oxidase on fabric properties Peroxidegeneration Carbohydrate Bleaching Final Time Glucose Oxidase NaOH FinalLiquor CIE Absorbency Sample # (min) (g/l) (U/ml) (g/l) pH color WI(second) S1 60 5 0.5 3 10.11 Light 30.49 <1 brown S2 60 5 1.5 3 11.13Light 36.46 <1 brown S3 240 5 0.5 3 10.32 Light 32.08 <1 brown S4 240 51.5 3 11.20 Light 38.26 <1 brown

Example 3 Effect of NaOH on Whiteness and Water Absorbency

All materials and chemicals were essentially the same as in Example 1.The peroxide generation and bleach experimental were the same as inExample 1 except that the amount of Carbohydrate Oxidase, glucose andsodium hydroxide varied. The value of whiteness index and absorbency ofcotton fabric were measured using the same methods in Example 1.

Table 3 shows the value of whiteness index and absorbency results.Higher absorbency is indicated by lower wetting time. Sodium hydroxidehas a positive impact on water absorbency. Increasing the concentrationsof carbohydrate oxidase and sodium hydroxide results in increase inwhiteness index of cotton fabric. Final liquor pH is the result fromNaOH addition. Higher final liquor pH is positively correlated to higherwater absorbency and higher whiteness of cotton fabric. TABLE 3 Effectof Carbohydrate Oxidase and NaOH Peroxide generation Sam- CarbohydrateBleaching ple Oxidase Glucose NaOH Final Absorbency CIE # (U/ml) (g/l)(g/l) pH (Second) WI S1 0.1 5 0 6.73 4.4(3-5) 27.47 S2 0.1 5 2 8.00 N/A*23.73 S3 0.1 5 4 11.69 <1 34.79 S4 0.1 5 8 12.37 <1 42.62 S5 0.5 5 06.34 6.0(5-7) 26.38 S6 0.5 5 2 8.12 1.4(1-2) 29.95 S7 0.5 5 4 11.80 <142.27 S8 0.5 5 8 12.40 <1 48.35 S9 1.5 5 0 5.14 4.0(3-6) 20.06 S10 1.5 52 8.45 1.2(1-2) 26.85 S11 1.5 5 4 11.90 <1 42.27 S12 1.5 5 8 12.45 <153.38*N/A: not measured

Example 4 Effect of NaOH and Silicate on Whiteness

All materials and chemicals were essentially the same as in Example 1.The peroxide generation and bleach experimental were the same as inExample 1 except that the amount of carbohydrate oxidase, glucose andsodium hydroxide varied. The value of whiteness index and absorbency ofcotton fabric were measured using the same methods in Example 1.

Table 4 shows the value of whiteness index and absorbency results.Increase NaOH dose results in increase in absorbency and whiteness ofcotton fabric. An optimal pH is about 12.2 in this experiment. Additionof silicate results in increase of fabric whiteness. TABLE 4 Impact ofNaOH and silicate on fabric properties Peroxide generation CarbohydrateBleaching Sample Oxidase Glucose Silicate NaOH Absorbency CIE # (U/ml)(g/l) (g/l) (g) Final pH (second) WI S1 3 6 0 0 4.3 1.3(1-2) 19.32 S2 36 3 0 5.8 1.0 34.67 S3 3 6 0 1 6.9 <1 27.24 S4 3 6 3 1 7.7 <1 42.57 S5 36 0 2 8.8 <1 36.69 S6 3 6 3 2 9.0 <1 39.17 S7 3 6 0 3 11.2 <1 46.09 S8 36 3 3 11.1 <1 51.62 S11 3 6 0 6 12.0 <1 59.14 S12 3 6 3 6 12.0 <1 65.70S13 3 6 0 8 12.2 <1 65.65 S14 3 6 3 8 12.2 <1 70.31 S15 3 6 0 10 12.4 <160.63 S16 3 6 3 10 12.4 <1 68.41

Example 5 Substrate Specificity of Carbohydrate Oxidase Base on the SameWeight

All materials and chemicals were essentially the same as in Example 1.The peroxide generation and bleach experimental were the same as inExample 1 except that the amount of Carbohydrate Oxidase, glucose andsodium hydroxide varied. The value of whiteness index and absorbency ofcotton fabric were measured using the same methods in Example 1. Afterbleaching, liquor pH was measured and liquor color was also observed andrecorded.

Table 5 shows the value of whiteness index and absorbency results. Basedon the same weight of substrate, the specific activity of CarbohydrateOxidase is ranked from high to low as:a-glucose>xylose>cellobiose>maltose>arabinose>galactose>fructose>mannose.All sugar tested in this study can be the substrate of CarbohydrateOxidase. TABLE 5 Substrate specificity of Carbohydrate Oxidase based onweight Peroxide generation Carbohydrate Bleaching Oxidase Sugar SugarNaOH Final Color of Sample # (U/ml) (6 g/l) (mmol) (g/l) pH Solution CIEWI S1 3 arabinose 40 8 12.2 Brown 40.06 S2 0 arabinose 40 8 12.2 Brown31.19 S3 3 xylose 40 8 12.3 Yellow 50.56 S4 0 xylose 40 8 12.2 Brown32.28 alpha- S5 3 glucose 33 8 12.3 Yellow 59.60 alpha- S6 0 glucose 338 12.2 Brown 34.80 Medium S7 3 galactose 33 8 12.2 brown 43.73 S8 0galactose 33 8 12.2 Brown 39.14 S9 3 fructose 33 8 12.2 Brown 38.65 S100 fructose 33 8 12.2 Brown 36.07 S11 3 mannose 33 8 12.2 Brown 37.95 S120 mannose 33 8 12.2 Brown 36.97 S13 3 cellobiose 18 8 12.3 Yellow 52.89S14 0 cellobiose 18 8 12.2 Brown 36.30 S15 3 maltose 17 8 12.3 Yellow51.92 S16 0 maltose 17 8 12.2 Brown 38.41

Example 6 Substrate Specificity of Carbohydrate Oxidase Base on the SameMolarity

All materials and chemicals were essentially the same as in Example 1.The peroxide generation and bleach experimental were the same as inExample 1 except that the amount of carbohydrate oxidase, glucose andsodium hydroxide varied. The value of whiteness index and absorbency ofcotton fabric were measured using the same methods in Example 1. Afterbleaching, liquor pH was measured and liquor color was also observed andrecorded.

Table 6 shows the value of whiteness index and absorbency results.Except dextrin, all other sugars are suitable to be substrates ofcarbohydrate oxidase. Since dextrin is the only polymer used here,dextrin of 1120 mg was used in each beaker for 14 gram fabric bleach.Based on the same mole of sugar, the substrate specificity ofcarbohydrate oxidase is ranked as:alpha-glucose>beta-glucose>xylose=beta-lactose>cellubiose=maltotriose>maltose.TABLE 6 Substrate specificity of Carbohydrate Oxidase based on the samemolarity Peroxide generation Carbohydrate Bleaching Oxidase Sugar NaOHFinal Color of Absorbency Sample # (U/ml) (22 mM) (g/l) pH Solution CIEWI (second) S1 3 Xylose 8 12.1 yellow 52.56 <1 S2 0 Xylose 8 12.1yellow+ 43.72 <1 S3 3 Beta- 8 12.1 yellow 55.48 <1 glucose S4 0 Beta- 812.1 yellow+ 44.17 <1 glucose S5 3 Alpha- 8 12.2 yellow 58.64 <1 glucoseS6 0 Alpha- 8 12.1 yellow+ 48.03 <1 glucose S7 3 cellobiose 8 12.1yellow 50.34 <1 S8 0 cellobiose 8 12.0 brown 35.52 <1 S9 3 maltose 812.0 brown 47.84 <1 S10 0 maltose 8 12.1 yellow 29.23 <1 S11 3maltotriose 8 11.8 brown+ 50.33 <1 S12 0 maltotriose 8 12.1 yellow 35.61<1 S13 3 Beta- 8 12.2 yellow 53.64 <1 lactose S14 0 Beta- 8 12.0 brown37.38 <1 lactose S15 3 Dextrin 8 12.2 yellow 50.38 <1 S16 0 Dextrin 812.2 yellow 50.89 <1

Example 7 Cotton Bleaching with Amylase and Carbohydrate Oxidase

A 100% cotton woven fabric 428R (TestFabrics) contains starch as sizingcomponent and has not been chemically treated after weaving. A 12 gswatch was treated in a Labomat (Mathis) beaker containing 120 mlsolution at each condition. The solution contained 0.5 g/l Kierlon jetB. 20 mM sodium phosphate buffer pH 7.0, 0.3 g/l calcium chloridedehydrate (Fisher Scientific), 2 g/l alpha-amylase AQUAZYM™ 240L(Novozymes North America, Inc.), and in some cases, 82 mg/l glucoamylaseSPIRIZYME™ PLUS FG (Novozymes North America, Inc). The treatment wasconducted at 50 rpm, 50° C. for 60 minutes. Then 1.7 ml/beaker of 0.21g/l silicate was added, and pH was adjusted with 3.3 ml/beaker of 0.3g/ml NaOH solution. The beaker was heated to 95° C. at 3° C./min and thetemperature was kept for 60 minutes. Swatches were then rinsed anddried.

The fabric whiteness index was measured in the same way as in Example 1.Combined alpha-amylase with carbohydrate oxidase treatment improvescotton fabric whiteness significantly compared to alpha-amylasetreatment alone. Addition of glucoamylase further improves fabricwhiteness. TABLE 7 Carbo- hydrate Whiteness Sample AQUAZYME ™SPIRIZYME ™ Oxidase Index # (g/l) Plus (mg/l) (U/ml) (Ganz 82) 1 2 0 018.6 2 2 0 0.25 45.0 3 2 0 3 46.8 4 2 82 3 51.1

Example 8

Measurement of the Activity of Fatty Acid Oxidizing Enzymes on LinoleicAcid

An “Oxi 3000 Oximeter” (WTW, Weilheim, Germany) with a TriOxmatic 300oxygen electrode and a standard reaction volume of 4 ml was used.

10 mg linoleic acid (10 ml 60% linoleic acid) was dissolved in 1 mlethanol, and 2 microliters Tween 20 was added. From this stock substratesolution 50 microliters was added into a reaction beaker containing 3.85ml buffer solution (Britton-Robinson: 100 mM of Phosphoric-, Acetic- andBoric acid; pH adjusted with NaOH) with a small stir bar allowing thesolution to be mixed well, and the oxygen electrode was inserted intothe reaction beaker. 100 microliters purified enzyme solution was added,viz. (a) lipoxygenase derived from Magnaporthe salvinii at aconcentration of approx. 0.4 mg/mL; or (b) lipoxygenase derived fromGaeumannomyces. graminis at a concentration of approx. 0.76 mg/ml (whichmeans approximately 0.02 mg/mL in the final reaction). Theselipoxygenases were prepared as previously described. The temperature was25° C. The concentration of dissolved oxygen (mg/l) is measured andplotted as a function of time (min.). The enzymatic activity iscalculated as the slope of the linear part of the curve (mg/l/min.)after addition of the enzyme. The baseline was corrected by subtractionwhen relevant, meaning that if the curve showing oxygen concentration asa function of time had a slope of above about 0.05 mg oxygen/m/minbefore addition of the fatty acid oxidizing enzyme (i.e. the control),this value was subtracted from the sample slope value.

Table 8 below shows the results of the experiments. TABLE 8 Fatty AcidOxidizing Enzyme (a) LOX from (b) LOX from M. salvinii mg G. graminis mgpH O₂/mL/min O₂/mL/min 2 0.0 0.0 4 0.4 0.1 5 0.7 0.4 6 1.1 0.4 7 1.0 0.48 0.7 0.5 9 0.8 0.4 10 0.7 0.4 11 0.6 0.2

Example 9

Fatty Acid Oxidizing Enzymes

Four enzymes, viz. two laccases and two lipoxygenases were tested asdescribed below. The laccase derived from Polyporus pinsitus had a MW bySDS-Page of 65 kDa, a pl by IEF of 3.5, and an optimum temperature at pH5.5 of 60° C. The laccase derived from Coprinus cinereus had a MW bySDS-Page of 67-68 kDa, a pl by IEF of 3.5-3.8, and an optimumtemperature at pH 7.5 of 65° C. The enzymes were prepared and purifiedas described in WO 96/00290 and U.S. Pat. No. 6,008,029. The twolipoxygenases were derived from Magnaporthe salvinii and Gaeumannomycesgraminis, and they were prepared as described previously.

The enzyme dosage was adjusted to ensure, maximum absorbancy increaseper minute at 234 nm/530 nm, viz in the range of 0.1-0.25 absorbancyunits per minute.

Substrate solution: 11.65 mg linoleic acid (60% Sigma), as well as 12.5ml 0.56 mM Syringaldazine (Sigma) in ethanol was mixed with deionizedwater to a total volume of 25 ml.

50 microliters of the enzyme preparation to be tested was transferred toa quartz cuvette containing 900 microliters phosphate buffer (50 mM, pH7.0) and 50 microliters of the substrate solution. The cuvette wasplaced in a spectrofotometer, thermostated at 23° C., and theabsorbancies at 234 nm and 530 nm were measured as a function of time.The absorbancy at 530 nm is indicative of degradation of syringaldazine,whereas the absorbancy at 234 nm is indicative of degradation oflinoleic acid. The absorbancy increase as a function of time iscalculated on the basis of minutes 2 to 4 of the reaction time, i.e.,d(A₂₃₄)/dt, as well as d(A₅₃₀)/dt.

The results are shown in Table 9 below. Of these four enzymes, only thetwo lipoxygenases qualify as a fatty acid oxidizing enzyme as definedherein. This is because RRD=Reaction Rate Difference=(dA₂₃₄/dt−dA₅₃₀/dt)is above zero only for these two enzymes. TABLE 9 dA₂₃₄/dt − dA₅₃₀/dtdA₂₃₄/dt dA₅₃₀/dt Enzyme (units/min) (units/min) (units/min) Polyporuspinsitus  0.20  0.002* −0.20 laccase Magnaporthe salvinii  0.0001*  0.130.13 lipoxygenase Coprinus cinereus  0.17 −0.001* −0.17 laccaseGaeumannomyces −0.03*  0.21 0.21 graminis lipoxygenase*this is equivalent to zero activity (analytical inaccuracy)

Example 10

Lipoxygenase (LOX) Bleaching of Cotton

Cotton fabric is 100% cotton interlock knit 4600 (Ramseur InterlockKnit, Inc., NC). The cotton fabric was cut into 19×19 cm² swatches(about 6.0 g per swatch).

Lipoxygenase from Magnaporthe salvinii was cloned and expressed inAspergillus oryzae as described in Example 2 of WO 02/086114. The enzymewas purified and stored at −18° C. prior to application.

Buffer A (50 mM) was made by dissolving 26.95 g Na₂HPO₄ 7H₂O in 2 litersdeionized water, and the pH was adjusted to 7 with 5 M HCl. Buffer B (50mM) was made by dissolving 38.23 g Na₂B₄O₇ 10H₂O in 2 liters deionizedwater, the pH was adjusted to 9.5 with 30% NaOH. About 1 g Kieralon JetB (0.5 g/l) was added in each buffer solution.

During the experiment, 120 ml buffer was added to each beaker containinga fabric swatch. Linoleic acid (99%, batch 71k2050) and lipoxygeneasewere then added sequentially into each beaker. Beakers were then sealedand installed in Labomat equipment (type BFA Beaker from Werner Mathis,N.C.). The treatment was conducted at 50 rpm, 50° C. (3° C./mingradient) for 120 minutes. All swatches were then taken out and rinsedwith cold water. The swatches were washed in a US-type washing machineat 40° C., cold rinsed twice, and then tumble dried for 50 minutes.Thereafter all swatches were equilibrated at 21° C. (70° F.) and 65%relative humidity for more than 24 hours. The wettability of theswatches was measured according to AATCC method 79 (incorporated byreference). The Fabric whiteness was measured using Macbeth Color-eye7000 spectrophotometer.

The result of this test is shown in Table 10 below and shows the fabricwhiteness after treatment at 50° C. for 2 hours. In the control with theabsence of both lipoxygenase (LOX) and lenoleic acid (LA), or theabsence of LA, fabric whiteness index changed very little in both pH 7.0and 9.5 treatments. In the presence of LOX and LA, fabric whitenessindex was significantly higher, indicating bleaching effect. Thebleaching was more effective at pH 7 than pH 9.5. TABLE 10 Treatingconditions Whiteness Lenoleic Index (WI Weight Wetting Lipoxygenase acidCIE Hunter Loss (sec.) Swatch # pH U/ml mL/mL Ganz82 60 (%) AverageSTDEV 1 7 0 0 24.1 58.1 3.1 >60 0.0 2 7 4.8 0 24.4 58.3 3.1 >60 0.0 3 74.8 3.3 × 10⁻³ 27.3 59.7 3.3 >60 0.0 4 9.5 0 0 23.7 57.9 3.0 >60 0.0 59.5 4.8 0 22.8 57.5 2.9 >60 0.0 6 9.5 4.8 3.3 × 10⁻³ 27.2 59.7 3.4 >600.0 Untreated 1.5 47.1 >180 0.0

Example 11

NaOH Enhanced Lipoxygenase (LOX) Bleaching of Cotton

The enzyme, fabric, and chemicals were the same as in Example 10.Buffers were pH 7 and pH 9.5 made the same way with the addition ofKieralon Jet B as in Example 10. The same protocol was conducted asdescribed in the experimental protocol of Example 10, except that aftertreatment at 50° C. for 120 minutes, NaOH (3 g/l) was added in eachbeaker and the beakers were heated to 95° C. (3° C./min gradient) andkept for 30 minutes. The fabric swatches were rinsed the same way as inExample 10. After equilibrating at 21° C. (70° F.) and 65% relativehumidity for more than 24 hours, the fabric swatches were analyzed asdone in Example 10.

The results of this example are shown Table 11 below. First all fabricswatches had substantially higher whiteness level than those in Example10 due to the removal of color impurities by NaOH boiling. Thereafter,in the presence of LOX and LA, fabric whiteness level (Ganz 82) reachedas high as about 53, which was significantly higher than any othercontrol. This higher whiteness level may be due to the activation ofhydroperoxide in alkaline medium. The table also shows that thelipoxygenase and linoleic acid system gives higher fabric weight lossand better fabric wettability than any control. TABLE 11 Treatingconditions Whiteness Lenoleic Index (WI Weight Wetting Lipoxygenase acidCIE Hunter Loss (sec.) Swatch # pH U/ml mL/mL Ganz82 60 (%) AverageSTDEV 1 7 0 0 41.7 67.1 5.4 2.7 1.2 2 7 4.8 0 41.0 66.8 5.3 3.8 0.8 3 74.8 3.3 × 10⁻³ 52.7 73.2 5.6 <1 0.0 4 9.5 0 0 30.3 61.2 4.4 30.0 13.4 59.5 4.8 0 30.0 61.1 4.4 18.0 4.6 6 9.5 4.8 3.3 × 10⁻³ 43.8 68.4 4.6 <10.0 Untreated 1.5 47.1 >180 0.0

Example 12

Lipoxygenase (LOX) Assisted Scouring of Cotton Knit

Cotton fabric is 100% cotton interlock knit 4600 (Ramseur InterlockKnit, Inc., NC). The cotton fabric was cut into 19.5×19.5 cm² swatches(about 7.0 g per swatch). Buffer (20 mM) was made by dissolving 22.86 gNa₂B₄O₇ 10H₂O in 3 liters deionized water, the pH was adjusted to 9.25with 30% NaOH. About 1.5 g Kieralon Jet B (0.5 g/l) was added in thebuffer solution.

Buffer 70 mL, pH 9.25, was added to each beaker containing a fabricswatch. Linoleic acid (L-2376, lot 072K1228) (0.4 ml/beaker), linolenicacid (L-2376, lot 072K1228) (5.7×10⁻³ mL/mL), pectate lyase (BIOPREP™3000L) (2.14 APSU/g fabric), and lipoxygenease from Magnaporthe salvinii(8.2 U/mL) were then added into each beaker. Beakers were then sealedand Installed In Labomat equipment (type BFA Beaker from Werner Mathis,N.C.). The treatment was conducted at 50 rpm, 50° C. (3° C./mingradient) for 30 minutes. 1 g/L Na₂CO₃H₂O (Aldrich) and 0.2 g/L sodiumethylenediamine tetracetate (EDTA) (Dexter Chemical) were added inLabomat beakers. The beakers were heated to 90° C. (3° C./min gradient)and kept for 10 minutes. The water in the beakers was drained aftercooled down to about 70° C. All swatches were then taken out and rinsedwith warm water (60° C.) for about 10 minutes and then washed in aUS-type washing machine at 40° C. and then cold rinsed twice. They werethen air dried.

After all swatches were equilibrated at 21° C. (70° F.) and 65% relativehumidity for at least 24 hours, their wettability was measured accordingto MTCC method 79 (hereby incorporated by reference). Fabric whitenesswas measured using Macbeth Color-eye 7000 spectrophotometer. Fabricwhiteness (CIE L*a*b* values and CIE Ganz 82) and fabric wettability(wetting time in seconds with ±standard deviation) are shown in Table 12below. Lipoxygenase was shown to enhance fabric wettability when usedtogether with pectate lyase. Regardless of the presence of pectatelyase, fabrics treated by lipoxygenase together with either linelonicacid (LNA) or linoleic acid (LA) have excellent wettability. Fabrictreated with lipoxygenase and linoleic add has higher whiteness. TABLE12 Wetting time Treatment L* a* b* CIE Ganz 82 (seconds) Control 88.10.7 9.6 25.9 >120 PAL + LOX 88.1 0.7 9.6 26.2 17.5(+/−2.4) LOX + LNA88.9 0.1 11.6 18.2 <1 PAL + LOX + LNA 88.8 0.1 11.6 18.1 <1 LOX + LA89.6 0.1 9.0 32.4 <1 PAL + LOX + LA 89.6 0.0 9.1 32.1 <1

Example 13 Lipoxygenase (LOX) for Scouring of Cotton Woven Fabric

Cotton fabric is chemically desized 100% cotton woven type 428U(Tesffabrics, Pa.). Cotton fabric was cut into 15×25.5 cm² swatches(about 9.0 g per swatch). The pectate lyase and the lipoxygenase werethe same as in Example 12. The cutinase was a variant of a cutinasederived from Humicola insolens, DSM1800 with activity of 17.2 KLU/g(batch PPW21399). Other chemicals and buffer were the same as in Example12.

Buffer 90 ml pH 9.25 was added to each beaker containing a fabricswatch. Linoleic acid (4.4×10⁻³ mL/mL), pectate lyase (2.14 APSU/gfabric), Cutinase (17 LU/g fabric), and lipoxygenease (6.4 U/mL) werethen added into each beaker. The experiment was conducted the same as inExample 12.

After all swatches were equilibrated at 21° C. (70° F.) and 65% relativehumidity for at least 24 hours, their wettability and whiteness weremeasured as described in Example 12. Prior to and after the treatment,fabric weight was obtained after equilibrated at 21° C. (70° F.), 65% RHfor at least 24 hours. The percent of weight loss was determined usingfollowing equation:Weight loss (%)=(Weight_(before)−Weight_(after))/Weight_(before)×100

Fabric whiteness (CIE L*a*b* values and CIE Ganz 82), weight loss (%),and wettability are shown in Table 13 below. Lipoxygenase treatmentgives higher percentage of fabric weight loss than control. Lipoxygenaseand linoleic acid treatment gives higher weight loss and improved fabricwhiteness compared to the control. The addition of lipoxygenasegenerates higher percentage of fabric weight loss than pectinasetreatment only. The addition of lipoxygenas and linoleic acid improvesfabric whiteness. TABLE 13 CIE Weight Wetting time Treatment L* a* b*Ganz 82 Loss (%) (Seconds) Control 84.7 1.8 9.4 18.7 1.20 <1 LOX 84.81.8 9.4 18.8 1.52 <1 LOX + LA 86.1 1.6 9.6 21.2 1.52 <1 PAL 84.8 1.8 9.319.3 1.24 <1 PAL + LOX 84.8 1.8 9.4 18.7 1.45 <1 PAL + LOX + LA 86.0 1.69.5 21.5 1.53 <1 PAL + CUT 85.0 1.7 9.2 20.3 1.67 <1 PAL + CUT + LOX84.9 1.8 9.3 19.8 1.32 <1 PAL + CUT + 86.7 1.5 9.2 24.3 1.47 <1 LOX + LALOX: lipoxygenase;LA: linoleic acid;PAL: pactate lyase;CUT: cutinase.

Example 14

Lipoxygenase and Amylase for Desizing and Scouring of Cotton

Cotton fabric is 100% cotton woven type 428R (Tesffabrcs, Pa.). It wascut into 15×15.2 cm² swatches (about 7.7 g per swatch) in this study.Lipoxygenase and cutinase were the same as In Example 11. Amylase isAQUAZYME™ ULTRA 1200L, a commercial product made by Novozymes A/S(Denmark). Other chemicals and buffer pH 7.0 were the same as in Example8 except buffer is 20 mM sodium phosphate.

About 77 ml buffer was added in each beaker that contains a fabricswatch. AQUAZYM™ Ultra was diluted 10× and then added 0.4 mL/beaker.Cutinase was added in a concentration of 11 LU/mL. Linoleic acid(5.2×10⁻³ mL/mL) and lipoxygenase (7.4 U/mL) were added. Desizing wascarried out at 70° C., 50 rpm for 30 minutes in a Labomat machine. Afterthe treatment, swatches were rinsed in hot water (60° C.) and cold water(25° C.) for 10 minutes each.

After all swatches were equilibrated at 21° C. (70° F.) and 65% relativehumidity for at least 24 hours, their wettability and whiteness weremeasured as described in Example 13. The starch residue on fabric wasevaluated in according with the TEGEWA violet scale (Textile PraxisInternational 1981(12), p. 9-11), where 1 is not desized, 9 iscompletely desized. Two lots of 100% cotton woven 428U (commerciallydesized from Tesffabrics) were rated 5.3 and 6.5.

Table 14 below shows fabric whiteness, wettability, and starch residuein TEGEWA scale. Lipoxygenase and linoleic acid improve fabric whitenessand wettability in either amylase desizing or amylase and cutinasedesizing. The addition of lipoxygenase to cutinase and amylase desizingsolution improves fabric wettability. TABLE 14 CIE Wetting time Name L*a* b* Ganz 82 (seconds) Tegewa Control 87.2 1.1 9.5 24.3  ≦1 1.0 AmL87.1 1.1 9.5 23.9 8.3(+/−1.0) 3.3 AmL + LOX 86.9 1.1 9.9 21.410.7(+/−0.5)  3.5 AmL + LOX + 87.7 0.9 10.0 22.9 1.0(+/−0.0) 2.8 LAAmL + Cut + 87.2 1.1 9.7 23.0 2.5(+/−0.5) 3.3 LOX Aml + Cut + 88.0 0.99.1 28.0 1.8(+/−0.8) 2.8 LOX + LA Untreated 86.3 1.3 11.3 13.1 >120 1.0LOX: lipoxygenase;LA: linoleic acid;AmL: Amylase;Cut: cutinase.

1. A method of treating textiles, in particular fabrics, fibers, oryarns comprising treating fabric, fiber, or yarn, in an aqueous medium,with a carbohydrate oxidase and/or a fatty acid oxidizing enzyme.
 2. Amethod of claim 1, comprising treating fabric, fiber, or yarn, in anaqueous medium, with an effective amount of a carbohydrate oxidasehaving activity towards monosaccharides and at least one ofdi-saccharides and oligo-saccharides, and a substrate for saidcarbohydrate oxidase.
 3. The method according to claim 1, wherein thefabric, fiber, or yarn is a cellulosic material.
 4. The method accordingto claim 3, wherein the cellulosic material is a cotton-containingmaterial.
 5. The method according to claims 1, wherein the carbohydrateoxidase is derived from fungi, from bacteria, or from algae.
 6. Themethod according to claim 5, wherein the carbohydrate oxidase is derivedfrom Microdochium.
 7. The method according to claim 6, wherein thecarbohydrate oxidase is derived from Microdochium nivale.
 8. The methodaccording to claim 1, wherein the concentration of the carbohydrateoxidase is in the range of from about 0.05 U/ml to about 10 U/ml. 9-10.(canceled)
 11. The method according to claim 1, wherein the carbohydratesubstrate is selected from the group consisting of alpha-glucose,beta-glucose, xylose, cellobiose, maltose, arabinose, galactose,fructose, maltriose, lactose, and mannose.
 12. The method according toclaim 1, wherein the concentration of the concentration of thecarbohydrate oxidase substrate is from about 1 to about 200 mM. 13-14.(canceled)
 15. The method according to claim 1, wherein the peroxidegenerating step is carried out at a pH in the range of about 5.5 toabout
 9. 16. (canceled)
 17. The method according to claim 1, wherein theaqueous medium is added a peroxide activator.
 18. The method accordingto claim 17, wherein the activator is silicate.
 19. The method accordingto claim 1, wherein the substrate is generated in situ with anotherenzyme or chemical system.
 20. The method according to claim 19, whereinthe enzyme system comprises at least one of the enzymes from the groupconsisting of cellulase, xylanase, mannanase, amylase, arabinase,galactase, pectinase and glucanase.
 21. A composition for use in amethod of treating fabrics, fibers, or yarns comprising a carbohydrateoxidase having activity towards monosaccharides and at least one ofdisaccharides and oligo-saccharides and a substrate for saidcarbohydrate oxidase.
 22. A method of claims 1, comprising a step oftreating the textile in an aqueous medium with one or more fatty acidoxidizing enzyme.
 23. The method of claim 22, wherein the treatment is ableaching step.
 24. The method of claim 23, wherein the bleaching stepis followed by an alkaline treatment step carried out at a pH above 8,preferably between 9 and
 13. 25. (canceled)
 26. The method of claim 22,wherein the treatment is a scouring step. 27-30. (canceled)
 31. Themethod of claims 22, wherein the treatment is a desizing step. 32-47.(canceled)
 48. A composition comprising a fatty acid oxidizing enzymeand in addition thereto at least one adjuvant, preferably a wettingagent, polymeric agent and/or dispersing agent.
 49. The composition ofclaim 48, wherein the fatty acid oxidizing enzyme is a lipoxygenase,preferably is derived from the genus Magnaporthe, especially a strain ofMagnaporthe salvinii.
 50. The composition of claim 48, wherein thecomposition further comprises an enzyme selected from the groupconsisting of: a proteolytic enzyme, a lipolytic enzyme, a cellulolyticenzyme, an amylolytic enzyme, a pectolytic enzyme, an oxidase enzyme, ora peroxidase enzyme, or mixtures hereof. 51-61. (canceled)